Genome sequencing detects a balanced pericentric inversion with breakpoints that impact the DMD and upstream region of POU3F4 genes.

We describe a family with two maternal half-brothers both of whom presented with muscular dystrophy, autism spectrum disorder, developmental delay, and sensorineural hearing loss. The elder brother had onset of features at ~3 months of age, followed by clinical confirmation of muscular dystrophy at 3 years. Skeletal biopsy staining at 4.7 years showed an absence of dystrophin protein which prompted extensive molecular testing over 4 years that included gene panels, targeted single-gene assays, arrays, and karyotyping, all of which failed to identify a clinically significant variant in the DMD gene. At 10 years of age, clinical whole-genome sequencing (cWGS) was performed, which revealed a novel hemizygous ~50.7 Mb balanced pericentric inversion on chromosome X that disrupts the DMD gene in both siblings, consistent with the muscular dystrophy phenotype. This inversion also impacts the upstream regulatory region of POU3F4, structural rearrangements which are known to cause hearing loss. The unaffected mother is a heterozygous carrier for the pericentric inversion. This finding illustrates the ability of cWGS to detect a wide breadth of disease-causing genomic variations including large genomic rearrangements.

Heterozygous NOTCH1 Variants Cause CNS Immune Activation and Microangiopathy.

NOTCH1 belongs to the NOTCH family of proteins that regulate cell fate and inflammatory responses. Somatic and germline NOTCH1 variants have been implicated in cancer, Adams-Oliver syndrome, and cardiovascular defects. We describe 7 unrelated patients grouped by the presence of leukoencephalopathy with calcifications and heterozygous de novo gain-of-function variants in NOTCH1. Immunologic profiling showed upregulated CSF IP-10, a cytokine secreted downstream of NOTCH1 signaling. Autopsy revealed extensive leukoencephalopathy and microangiopathy with vascular calcifications. This evidence implicates that heterozygous gain-of-function variants in NOTCH1 lead to a chronic central nervous system (CNS) inflammatory response resulting in a calcifying microangiopathy with leukoencephalopathy. ANN NEUROL 2022;92:895-901.

The smallest likely pathogenic duplication of a SOX9 enhancer identified to date in a family with 46,XX testicular differences of sex development.

Copy number variants that duplicate distal upstream enhancer elements of the SOX9 gene cause 46,XX testicular differences of sex development (DSD) which is characterized by a 46,XX karyotype in an individual presenting with either ambiguous genitalia or genitalia with varying degrees of virilization, including those resembling typical male genitalia. Reported duplications in this region range in size from 24 to 780 kilobases (kb). Here we report a family with two affected individuals, the proband and his maternal uncle, harboring a 3.7 kb duplication of a SOX9 enhancer identified by clinical genome sequencing. Prior fluorescence in situ hybridization (FISH) for SRY and a multi-gene panel for ambiguous genitalia were non-diagnostic. The unaffected mother also carries this duplication, consistent with previously described incomplete penetrance. To our knowledge, this is the smallest duplication identified to-date, most of which resides in a 5.2 kb region that has been previously shown to possess enhancer activity that promotes the expression of SOX9. The duplication was confirmed by quantitative-PCR and shown to be in tandem by bidirectional Sanger sequencing breakpoint analysis. This finding highlights the importance of non-coding variant interrogation in suspected genetic disorders.

mafba is a downstream transcriptional effector of Vegfc signaling essential for embryonic lymphangiogenesis in zebrafish.

The lymphatic vasculature plays roles in tissue fluid balance, immune cell trafficking, fatty acid absorption, cancer metastasis, and cardiovascular disease. Lymphatic vessels form by lymphangiogenesis, the sprouting of new lymphatics from pre-existing vessels, in both development and disease contexts. The apical signaling pathway in lymphangiogenesis is the VEGFC/VEGFR3 pathway, yet how signaling controls cellular transcriptional output remains unknown. We used a forward genetic screen in zebrafish to identify the transcription factor mafba as essential for lymphatic vessel development. We found that mafba is required for the migration of lymphatic precursors after their initial sprouting from the posterior cardinal vein. mafba expression is enriched in sprouts emerging from veins, and we show that mafba functions cell-autonomously during lymphatic vessel development. Mechanistically, Vegfc signaling increases mafba expression to control downstream transcription, and this regulatory relationship is dependent on the activity of SoxF transcription factors, which are essential for mafba expression in venous endothelium. Here we identify an indispensable Vegfc-SoxF-Mafba pathway in lymphatic development.

A clinical laboratory's experience using GeneMatcher-Building stronger gene-disease relationships.

The use of whole-genome sequencing (WGS) has accelerated the pace of gene discovery and highlighted the need for open and collaborative data sharing in the search for novel disease genes and variants. GeneMatcher (GM) is designed to facilitate connections between researchers, clinicians, health-care providers, and others to help in the identification of additional patients with variants in the same candidate disease genes. The Illumina Clinical Services Laboratory offers a WGS test for patients with suspected rare and undiagnosed genetic disease  and regularly submits potential candidate genes to GM to strengthen gene-disease relationships. We describe our experience with GM, including criteria for evaluation of candidate genes, and our workflow for the submission and review process. We have made 69 submissions, 36 of which are currently active. Ten percent of submissions have resulted in publications, with an additional 14 submissions part of ongoing collaborations and expected to result in a publication.

De novo mutations in TOMM70, a receptor of the mitochondrial import translocase, cause neurological impairment.

The translocase of outer mitochondrial membrane (TOMM) complex is the entry gate for virtually all mitochondrial proteins and is essential to build the mitochondrial proteome. TOMM70 is a receptor that assists mainly in mitochondrial protein import. Here, we report two individuals with de novo variants in the C-terminal region of TOMM70. While both individuals exhibited shared symptoms including hypotonia, hyper-reflexia, ataxia, dystonia and significant white matter abnormalities, there were differences between the two individuals, most prominently the age of symptom onset. Both individuals were undiagnosed despite extensive genetics workups. Individual 1 was found to have a p.Thr607Ile variant while Individual 2 was found to have a p.Ile554Phe variant in TOMM70. To functionally assess both TOMM70 variants, we replaced the Drosophila Tom70 coding region with a Kozak-mini-GAL4 transgene using CRISPR-Cas9. Homozygous mutant animals die as pupae, but lethality is rescued by the mini-GAL4-driven expression of human UAS-TOMM70 cDNA. Both modeled variants lead to significantly less rescue indicating that they are loss-of-function alleles. Similarly, RNAi-mediated knockdown of Tom70 in the developing eye causes roughening and synaptic transmission defect, common findings in neurodegenerative and mitochondrial disorders. These phenotypes were rescued by the reference, but not the variants, of TOMM70. Altogether, our data indicate that de novo loss-of-function variants in TOMM70 result in variable white matter disease and neurological phenotypes in affected individuals.

Heterozygous Variants in the Mechanosensitive Ion Channel TMEM63A Result in Transient Hypomyelination during Infancy.

Mechanically activated (MA) ion channels convert physical forces into electrical signals. Despite the importance of this function, the involvement of mechanosensitive ion channels in human disease is poorly understood. Here we report heterozygous missense mutations in the gene encoding the MA ion channel TMEM63A that result in an infantile disorder resembling a hypomyelinating leukodystrophy. Four unrelated individuals presented with congenital nystagmus, motor delay, and deficient myelination on serial scans in infancy, prompting the diagnosis of Pelizaeus-Merzbacher (like) disease. Genomic sequencing revealed that all four individuals carry heterozygous missense variants in the pore-forming domain of TMEM63A. These variants were confirmed to have arisen de novo in three of the four individuals. While the physiological role of TMEM63A is incompletely understood, it is highly expressed in oligodendrocytes and it has recently been shown to be a MA ion channel. Using patch clamp electrophysiology, we demonstrated that each of the modeled variants result in strongly attenuated stretch-activated currents when expressed in naive cells. Unexpectedly, the clinical evolution of all four individuals has been surprisingly favorable, with substantial improvements in neurological signs and developmental progression. In the three individuals with follow-up scans after 4 years of age, the myelin deficit had almost completely resolved. Our results suggest a previously unappreciated role for mechanosensitive ion channels in myelin development.

Bi-allelic CSF1R Mutations Cause Skeletal Dysplasia of Dysosteosclerosis-Pyle Disease Spectrum and Degenerative Encephalopathy with Brain Malformation.

Colony stimulating factor 1 receptor (CSF1R) plays key roles in regulating development and function of the monocyte/macrophage lineage, including microglia and osteoclasts. Mono-allelic mutations of CSF1R are known to cause hereditary diffuse leukoencephalopathy with spheroids (HDLS), an adult-onset progressive neurodegenerative disorder. Here, we report seven affected individuals from three unrelated families who had bi-allelic CSF1R mutations. In addition to early-onset HDLS-like neurological disorders, they had brain malformations and skeletal dysplasia compatible to dysosteosclerosis (DOS) or Pyle disease. We identified five CSF1R mutations that were homozygous or compound heterozygous in these affected individuals. Two of them were deep intronic mutations resulting in abnormal inclusion of intron sequences in the mRNA. Compared with Csf1r-null mice, the skeletal and neural phenotypes of the affected individuals appeared milder and variable, suggesting that at least one of the mutations in each affected individual is hypomorphic. Our results characterized a unique human skeletal phenotype caused by CSF1R deficiency and implied that bi-allelic CSF1R mutations cause a spectrum of neurological and skeletal disorders, probably depending on the residual CSF1R function.

PERCHING syndrome: Clinical presentation in the first African patient confirmed by clinical whole genome sequencing.

PERCHING syndrome is a rare multisystem developmental disorder caused by autosomal recessive (AR) variants (truncating and missense) in the Kelch-like family member 7 gene (KLHL7). We report the first phenotypic and molecular description of PERCHING syndrome in a patient from Central Africa. The patient presented multiple dysmorphic features in addition to neurological, respiratory, gastroenteric, and dysautonomic disorders. Clinical Whole Genome Sequencing in the proband and his mother identified two novel heterozygous variants in the KLHL7 gene, including a maternally inherited intronic variant (NM_001031710.2:c.793 + 5G > C) classified as Variant of Uncertain Significance and a frameshift stop gain variant (NM_001031710.2:c.944delG; p.Ser315ThrfsTer23) of unknown inheritance classified as likely pathogenic. Although the diagnosis was only evoked after genomic testing, the review of published patients suggests that this disease could be clinically recognizable and maybe considered as an encephalopathy. Our report will allow expanding the phenotypic and molecular spectrum of Perching syndrome.

Mutations in PIGS, Encoding a GPI Transamidase, Cause a Neurological Syndrome Ranging from Fetal Akinesia to Epileptic Encephalopathy.

Inherited GPI deficiencies (IGDs) are a subset of congenital disorders of glycosylation that are increasingly recognized as a result of advances in whole-exome sequencing (WES) and whole-genome sequencing (WGS). IGDs cause a series of overlapping phenotypes consisting of seizures, dysmorphic features, multiple congenital malformations, and severe intellectual disability. We present a study of six individuals from three unrelated families in which WES or WGS identified bi-allelic phosphatidylinositol glycan class S (PIGS) biosynthesis mutations. Phenotypes included severe global developmental delay, seizures (partly responding to pyridoxine), hypotonia, weakness, ataxia, and dysmorphic facial features. Two of them had compound-heterozygous variants c.108G>A (p.Trp36∗) and c.101T>C (p.Leu34Pro), and two siblings of another family were homozygous for a deletion and insertion leading to p.Thr439_Lys451delinsArgLeuLeu. The third family had two fetuses with multiple joint contractures consistent with fetal akinesia. They were compound heterozygous for c.923A>G (p.Glu308Gly) and c.468+1G>C, a splicing mutation. Flow-cytometry analyses demonstrated that the individuals with PIGS mutations show a GPI-AP deficiency profile. Expression of the p.Trp36∗ variant in PIGS-deficient HEK293 cells revealed only partial restoration of cell-surface GPI-APs. In terms of both biochemistry and phenotype, loss of function of PIGS shares features with PIGT deficiency and other IGDs. This study contributes to the understanding of the GPI-AP biosynthesis pathway by describing the consequences of PIGS disruption in humans and extending the family of IGDs.

Cyrius: accurate CYP2D6 genotyping using whole-genome sequencing data.

Responsible for the metabolism of ~21% of clinically used drugs, CYP2D6 is a critical component of personalized medicine initiatives. Genotyping CYP2D6 is challenging due to sequence similarity with its pseudogene paralog CYP2D7 and a high number and variety of common structural variants (SVs). Here we describe a novel bioinformatics method, Cyrius, that accurately genotypes CYP2D6 using whole-genome sequencing (WGS) data. We show that Cyrius has superior performance (96.5% concordance with truth genotypes) compared to existing methods (84-86.8%). After implementing the improvements identified from the comparison against the truth data, Cyrius's accuracy has since been improved to 99.3%. Using Cyrius, we built a haplotype frequency database from 2504 ethnically diverse samples and estimate that SV-containing star alleles are more frequent than previously reported. Cyrius will be an important tool to incorporate pharmacogenomics in WGS-based precision medicine initiatives.

Expanded phenotype of AARS1-related white matter disease.

PURPOSE: Recent reports of individuals with cytoplasmic transfer RNA (tRNA) synthetase-related disorders have identified cases with phenotypic variability from the index presentations. We sought to assess phenotypic variability in individuals with AARS1-related disease. METHODS: A cross-sectional survey was performed on individuals with biallelic variants in AARS1. Clinical data, neuroimaging, and genetic testing results were reviewed. Alanyl tRNA synthetase (AlaRS) activity was measured in available fibroblasts. RESULTS: We identified 11 affected individuals. Two phenotypic presentations emerged, one with early infantile-onset disease resembling the index cases of AARS1-related epileptic encephalopathy with deficient myelination (n = 7). The second (n = 4) was a later-onset disorder, where disease onset occurred after the first year of life and was characterized on neuroimaging by a progressive posterior predominant leukoencephalopathy evolving to include the frontal white matter. AlaRS activity was significantly reduced in five affected individuals with both early infantile-onset and late-onset phenotypes. CONCLUSION: We suggest that variants in AARS1 result in a broader clinical spectrum than previously appreciated. The predominant form results in early infantile-onset disease with epileptic encephalopathy and deficient myelination. However, a subgroup of affected individuals manifests with late-onset disease and similarly rapid progressive clinical decline. Longitudinal imaging and clinical follow-up will be valuable in understanding factors affecting disease progression and outcome.

Randomized Clinical Trial of First-Line Genome Sequencing in Pediatric White Matter Disorders.

OBJECTIVE: Genome sequencing (GS) is promising for unsolved leukodystrophies, but its efficacy has not been prospectively studied. METHODS: A prospective time-delayed crossover design trial of GS to assess the efficacy of GS as a first-line diagnostic tool for genetic white matter disorders took place between December 1, 2015 and September 27, 2017. Patients were randomized to receive GS immediately with concurrent standard of care (SoC) testing, or to receive SoC testing for 4 months followed by GS. RESULTS: Thirty-four individuals were assessed at interim review. The genetic origin of 2 patient's leukoencephalopathy was resolved before randomization. Nine patients were stratified to the immediate intervention group and 23 patients to the delayed-GS arm. The efficacy of GS was significant relative to SoC in the immediate (5/9 [56%] vs 0/9 [0%]; Wild-Seber, p < 0.005) and delayed (control) arms (14/23 [61%] vs 5/23 [22%]; Wild-Seber, p < 0.005). The time to diagnosis was significantly shorter in the immediate-GS group (log-rank test, p = 0.04). The overall diagnostic efficacy of combined GS and SoC approaches was 26 of 34 (76.5%, 95% confidence interval = 58.8-89.3%) in <4 months, greater than historical norms of <50% over 5 years. Owing to loss of clinical equipoise, the trial design was altered to a single-arm observational study. INTERPRETATION: In this study, first-line GS provided earlier and greater diagnostic efficacy in white matter disorders. We provide an evidence-based diagnostic testing algorithm to enable appropriate clinical GS utilization in this population. ANN NEUROL 2020;88:264-273.

Biallelic ASCC1 variants including a novel intronic variant result in expanded phenotypic spectrum of spinal muscular atrophy with congenital bone fractures 2 (SMABF2).

Spinal muscular atrophy with congenital bone fractures 2 (SMABF2), a type of arthrogryposis multiplex congenita (AMC), is characterized by congenital joint contractures, prenatal fractures of long bones, and respiratory distress and results from biallelic variants in ASCC1. Here, we describe an infant with severe, diffuse hypotonia, congenital contractures, and pulmonary hypoplasia characteristic of SMABF2, with the unique features of cleft palate, small spleen, transverse liver, and pulmonary thromboemboli with chondroid appearance. This infant also had impaired coagulation with diffuse petechiae and ecchymoses which has only been reported in one other infant with AMC. Using trio whole genome sequencing, our proband was identified to have biallelic variants in ASCC1. Using deep next generation sequencing of parental cDNA, we characterized alteration of splicing encoded by the novel, maternally inherited ASCC1 variant (c.297-8 T > G) which provides a mechanism for functional pathogenicity. The paternally inherited ASCC1 variant is a rare nonsense variant (c.466C > T; p.Arg156*) that has been previously identified in one other infant with AMC. This report extends the phenotypic characteristics of ASCC1-associated AMC (SMABF2) and describes a novel intronic variant that partially disrupts RNA splicing.

Detection of long repeat expansions from PCR-free whole-genome sequence data.

Identifying large expansions of short tandem repeats (STRs), such as those that cause amyotrophic lateral sclerosis (ALS) and fragile X syndrome, is challenging for short-read whole-genome sequencing (WGS) data. A solution to this problem is an important step toward integrating WGS into precision medicine. We developed a software tool called ExpansionHunter that, using PCR-free WGS short-read data, can genotype repeats at the locus of interest, even if the expanded repeat is larger than the read length. We applied our algorithm to WGS data from 3001 ALS patients who have been tested for the presence of the C9orf72 repeat expansion with repeat-primed PCR (RP-PCR). Compared against this truth data, ExpansionHunter correctly classified all (212/212, 95% CI [0.98, 1.00]) of the expanded samples as either expansions (208) or potential expansions (4). Additionally, 99.9% (2786/2789, 95% CI [0.997, 1.00]) of the wild-type samples were correctly classified as wild type by this method with the remaining three samples identified as possible expansions. We further applied our algorithm to a set of 152 samples in which every sample had one of eight different pathogenic repeat expansions, including those associated with fragile X syndrome, Friedreich's ataxia, and Huntington's disease, and correctly flagged all but one of the known repeat expansions. Thus, ExpansionHunter can be used to accurately detect known pathogenic repeat expansions and provides researchers with a tool that can be used to identify new pathogenic repeat expansions.

Identification of miR-29b targets using 3-cyanovinylcarbazole containing mimics.

MicroRNAs (miRNAs) are highly conserved ∼22 nt small noncoding RNAs that bind partially complementary sequences in target transcripts. MicroRNAs regulate both translation and transcript stability, and play important roles in development, cellular homeostasis, and disease. There are limited approaches available to agnostically identify microRNA targets transcriptome-wide, and methods using miRNA mimics, which in principle identify direct miRNA:transcript pairs, have low sensitivity and specificity. Here, we describe a novel method to identify microRNA targets using miR-29b mimics containing 3-cyanovinylcarbazole (CNVK), a photolabile nucleoside analog. We demonstrate that biotin-tagged, CNVK-containing miR-29b (CNVK-miR-29b) mimics are nontoxic in cell culture, associate with endogenous mammalian Argonaute2, are sensitive for known targets and recapitulate endogenous transcript destabilization. Partnering CNVK-miR-29b with ultra-low-input RNA sequencing, we recover ∼40% of known miR-29b targets and find conservation of the focal adhesion and apoptotic target pathways in mouse and human. We also identify hundreds of novel targets, including NRAS, HOXA10, and KLF11, with a validation rate of 71% for a subset of 73 novel target transcripts interrogated using a high-throughput luciferase assay. Consistent with previous reports, we show that both endogenous miR-29b and CNVK-miR-29b are trafficked to the nucleus, but find no evidence of nuclear-specific miR-29b transcript binding. This may indicate that miR-29b nuclear sequestration is a regulatory mechanism in itself. We suggest that CNVK-containing small RNA mimics may find applicability in other experimental models.

ExpansionHunter: a sequence-graph-based tool to analyze variation in short tandem repeat regions.

SUMMARY: We describe a novel computational method for genotyping repeats using sequence graphs. This method addresses the long-standing need to accurately genotype medically important loci containing repeats adjacent to other variants or imperfect DNA repeats such as polyalanine repeats. Here we introduce a new version of our repeat genotyping software, ExpansionHunter, that uses this method to perform targeted genotyping of a broad class of such loci. AVAILABILITY AND IMPLEMENTATION: ExpansionHunter is implemented in C++ and is available under the Apache License Version 2.0. The source code, documentation, and Linux/macOS binaries are available at https://github.com/Illumina/ExpansionHunter/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Spinal muscular atrophy diagnosis and carrier screening from genome sequencing data.

PURPOSE: Spinal muscular atrophy (SMA), caused by loss of the SMN1 gene, is a leading cause of early childhood death. Due to the near identical sequences of SMN1 and SMN2, analysis of this region is challenging. Population-wide SMA screening to quantify the SMN1 copy number (CN) is recommended by the American College of Medical Genetics and Genomics. METHODS: We developed a method that accurately identifies the CN of SMN1 and SMN2 using genome sequencing (GS) data by analyzing read depth and eight informative reference genome differences between SMN1/2. RESULTS: We characterized SMN1/2 in 12,747 genomes, identified 1568 samples with SMN1 gains or losses and 6615 samples with SMN2 gains or losses, and calculated a pan-ethnic carrier frequency of 2%, consistent with previous studies. Additionally, 99.8% of our SMN1 and 99.7% of SMN2 CN calls agreed with orthogonal methods, with a recall of 100% for SMA and 97.8% for carriers, and a precision of 100% for both SMA and carriers. CONCLUSION: This SMN copy-number caller can be used to identify both carrier and affected status of SMA, enabling SMA testing to be offered as a comprehensive test in neonatal care and an accurate carrier screening tool in GS sequencing projects.

Case for genome sequencing in infants and children with rare, undiagnosed or genetic diseases.

Up to 350 million people worldwide suffer from a rare disease, and while the individual diseases are rare, in aggregate they represent a substantial challenge to global health systems. The majority of rare disorders are genetic in origin, with children under the age of five disproportionately affected. As these conditions are difficult to identify clinically, genetic and genomic testing have become the backbone of diagnostic testing in this population. In the last 10 years, next-generation sequencing technologies have enabled testing of multiple disease genes simultaneously, ranging from targeted gene panels to exome sequencing (ES) and genome sequencing (GS). GS is quickly becoming a practical first-tier test, as cost decreases and performance improves. A growing number of studies demonstrate that GS can detect an unparalleled range of pathogenic abnormalities in a single laboratory workflow. GS has the potential to deliver unbiased, rapid and accurate molecular diagnoses to patients across diverse clinical indications and complex presentations. In this paper, we discuss clinical indications for testing and historical testing paradigms. Evidence supporting GS as a diagnostic tool is supported by superior genomic coverage, types of pathogenic variants detected, simpler laboratory workflow enabling shorter turnaround times, diagnostic and reanalysis yield, and impact on healthcare.

Correction: Estimating the burden and economic impact of pediatric genetic disease.

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