Michael Yourshaw

Clinical Exome Sequencing for Genetic Identification of Rare Mendelian Disorders

IMPORTANCE Clinical exome sequencing (CES) is rapidly becoming a common molecular diagnostic test for individuals with rare genetic disorders. OBJECTIVE To report on initial clinical indications for CES referrals and molecular diagnostic rates for different indications and for different test types. DESIGN, SETTING, AND PARTICIPANTS Clinical exome sequencing was performed on 814 consecutive patients with undiagnosed, suspected genetic conditions at the University of California, Los Angeles, Clinical Genomics Center between January 2012 and August 2014. Clinical exome sequencing was conducted as trio-CES (both parents and their affected child sequenced simultaneously) to effectively detect de novo and compound heterozygous variants or as proband-CES (only the affected individual sequenced) when parental samples were not available. MAIN OUTCOMES AND MEASURES Clinical indications for CES requests, molecular diagnostic rates of CES overall and for phenotypic subgroups, and differences in molecular diagnostic rates between trio-CES and proband-CES. RESULTS Of the 814 cases, the overall molecular diagnosis rate was 26% (213 of 814; 95% CI, 23%-29%). The molecular diagnosis rate for trio-CES was 31% (127 of 410 cases; 95% CI, 27%-36%) and 22% (74 of 338 cases; 95% CI, 18%-27%) for proband-CES. In cases of developmental delay in children (<5 years, n = 138), the molecular diagnosis rate was 41% (45 of 109; 95% CI, 32%-51%) for trio-CES cases and 9% (2 of 23, 95% CI, 1%-28%) for proband-CES cases. The significantly higher diagnostic yield (P value = .002; odds ratio, 7.4 [95% CI, 1.6-33.1]) of trio-CES was due to the identification of de novo and compound heterozygous variants. CONCLUSIONS AND RELEVANCE In this sample of patients with undiagnosed, suspected genetic conditions, trio-CES was associated with higher molecular diagnostic yield than proband-CES or traditional molecular diagnostic methods. Additional studies designed to validate these findings and to explore the effect of this approach on clinical and economic outcomes are warranted.

Mutations in the RNA exosome component gene EXOSC3 cause pontocerebellar hypoplasia and spinal motor neuron degeneration

RNA exosomes are multi-subunit complexes conserved throughout evolution and are emerging as the major cellular machinery for processing, surveillance and turnover of a diverse spectrum of coding and noncoding RNA substrates essential for viability. By exome sequencing, we discovered recessive mutations in EXOSC3 (encoding exosome component 3) in four siblings with infantile spinal motor neuron disease, cerebellar atrophy, progressive microcephaly and profound global developmental delay, consistent with pontocerebellar hypoplasia type 1 (PCH1; MIM 607596). We identified mutations in EXOSC3 in an additional 8 of 12 families with PCH1. Morpholino knockdown of exosc3 in zebrafish embryos caused embryonic maldevelopment, resulting in small brain size and poor motility, reminiscent of human clinical features, and these defects were largely rescued by co-injection with wild-type but not mutant exosc3 mRNA. These findings represent the first example of an RNA exosome core component gene that is responsible for a human disease and further implicate dysregulation of RNA processing in cerebellar and spinal motor neuron maldevelopment and degeneration.

Pontocerebellar hypoplasia type 1 Clinical spectrum and relevance of EXOSC3 mutations

Objectives: Pontocerebellar hypoplasia with spinal muscular atrophy, also known as PCH1, is a group of autosomal recessive disorders characterized by generalized muscle weakness and global developmental delay commonly resulting in early death. Gene defects had been discovered only in single patients until the recent identification of EXOSC3 mutations in several families with relatively mild course of PCH1. We aim to genetically stratify subjects in a large and well-defined cohort to define the clinical spectrum and genotype–phenotype correlation. Methods: We documented clinical, neuroimaging, and morphologic data of 37 subjects from 27 families with PCH1. EXOSC3 gene sequencing was performed in 27 unrelated index patients of mixed ethnicity. Results: Biallelic mutations in EXOSC3 were detected in 10 of 27 families (37%). The most common mutation among all ethnic groups was c.395A>C, p.D132A, responsible for 11 (55%) of the 20 mutated alleles and ancestral in origin. The mutation-positive subjects typically presented with normal pregnancy, normal birth measurements, and relative preservation of brainstem and cortical structures. Psychomotor retardation was profound in all patients but lifespan was variable, with 3 subjects surviving beyond the late teens. Abnormal oculomotor function was commonly observed in patients surviving beyond the first year. Major clinical features previously reported in PCH1, including intrauterine abnormalities, postnatal hypoventilation and feeding difficulties, joint contractures, and neonatal death, were rarely observed in mutation-positive infants but were typical among the mutation-negative subjects. Conclusion: EXOSC3 mutations account for 30%–40% of patients with PCH1 with variability in survival and clinical severity that is correlated with the genotype.

Rich annotation of DNA sequencing variants by leveraging the Ensembl Variant Effect Predictor with plugins

High-throughput DNA sequencing has become a mainstay for the discovery of genomic variants that may cause disease or affect phenotype. A next-generation sequencing pipeline typically identifies thousands of variants in each sample. A particular challenge is the annotation of each variant in a way that is useful to downstream consumers of the data, such as clinical sequencing centers or researchers. These users may require that all data storage and analysis remain on secure local servers to protect patient confidentiality or intellectual property, may have unique and changing needs to draw on a variety of annotation data sets and may prefer not to rely on closed-source applications beyond their control. Here we describe scalable methods for using the plugin capability of the Ensembl Variant Effect Predictor to enrich its basic set of variant annotations with additional data on genes, function, conservation, expression, diseases, pathways and protein structure, and describe an extensible framework for easily adding additional custom data sets.

Rare Genomic Variants Link Bipolar Disorder with Anxiety Disorders to CREB-Regulated Intracellular Signaling Pathways

Bipolar disorder is a common, complex, and severe psychiatric disorder with cyclical disturbances of mood and a high suicide rate. Here, we describe a family with four siblings, three affected females and one unaffected male. The disease course was characterized by early-onset bipolar disorder and co-morbid anxiety spectrum disorders that followed the onset of bipolar disorder. Genetic risk factors were suggested by the early onset of the disease, the severe disease course, including multiple suicide attempts, and lack of adverse prenatal or early life events. In particular, drug and alcohol abuse did not contribute to the disease onset. Exome sequencing identified very rare, heterozygous, and likely protein-damaging variants in eight brain-expressed genes: IQUB, JMJD1C, GADD45A, GOLGB1, PLSCR5, VRK2, MESDC2, and FGGY. The variants were shared among all three affected family members but absent in the unaffected sibling and in more than 200 controls. The genes encode proteins with significant regulatory roles in the ERK/MAPK and CREB-regulated intracellular signaling pathways. These pathways are central to neuronal and synaptic plasticity, cognition, affect regulation and response to chronic stress. In addition, proteins in these pathways are the target of commonly used mood-stabilizing drugs, such as tricyclic antidepressants, lithium, and valproic acid. The combination of multiple rare, damaging mutations in these central pathways could lead to reduced resilience and increased vulnerability to stressful life events. Our results support a new model for psychiatric disorders, in which multiple rare, damaging mutations in genes functionally related to a common signaling pathway contribute to the manifestation of bipolar disorder.

Functional consequences of a novel variant of PCSK1.

Background Common single nucleotide polymorphisms (SNPs) in proprotein convertase subtilisin/kexin type 1 with modest effects on PC1/3 in vitro have been associated with obesity in five genome-wide association studies and with diabetes in one genome-wide association study. We here present a novel SNP and compare its biosynthesis, secretion and catalytic activity to wild-type enzyme and to SNPs that have been linked to obesity. Methodology/Principal Findings A novel PC1/3 variant introducing an Arg to Gln amino acid substitution at residue 80 (within the secondary cleavage site of the prodomain) (rs1799904) was studied. This novel variant was selected for analysis from the 1000 Genomes sequencing project based on its predicted deleterious effect on enzyme function and its comparatively more frequent allele frequency. The actual existence of the R80Q (rs1799904) variant was verified by Sanger sequencing. The effects of this novel variant on the biosynthesis, secretion, and catalytic activity were determined; the previously-described obesity risk SNPs N221D (rs6232), Q665E/S690T (rs6234/rs6235), and the Q665E and S690T SNPs (analyzed separately) were included for comparative purposes. The novel R80Q (rs1799904) variant described in this study resulted in significantly detrimental effects on both the maturation and in vitro catalytic activity of PC1/3. Conclusion/Significance Our findings that this novel R80Q (rs1799904) variant both exhibits adverse effects on PC1/3 activity and is prevalent in the population suggests that further biochemical and genetic analysis to assess its contribution to the risk of metabolic disease within the general population is warranted.

Exome sequencing finds a novel PCSK1 mutation in a child with generalized malabsorptive diarrhea and diabetes insipidus.

Objectives: Congenital diarrhea disorders are a group of genetically diverse and typically autosomal recessive disorders that have yet to be well characterized phenotypically or molecularly. Diagnostic assessments are generally limited to nutritional challenges and histologic evaluation, and many subjects eventually require a prolonged course of intravenous nutrition. Here we describe next-generation sequencing techniques to investigate a child with perplexing congenital malabsorptive diarrhea and other presumably unrelated clinical problems; this method provides an alternative approach to molecular diagnosis. Methods: We screened the diploid genome of an affected individual, using exome sequencing, for uncommon variants that have observed protein-coding consequences. We assessed the functional activity of the mutant protein, as well as its lack of expression using immunohistochemistry. Results: Among several rare variants detected was a homozygous nonsense mutation in the catalytic domain of the proprotein convertase subtilisin/kexin type 1 gene. The mutation abolishes prohormone convertase 1/3 endoprotease activity as well as expression in the intestine. These primary genetic findings prompted a careful endocrine reevaluation of the child at 4.5 years of age, and multiple significant problems were subsequently identified consistent with the known phenotypic consequences of proprotein convertase subtilisin/kexin type 1 (PCSK1) gene mutations. Based on the molecular diagnosis, alternate medical and dietary management was implemented for diabetes insipidus, polyphagia, and micropenis. Conclusions: Whole-exome sequencing provides a powerful diagnostic tool to clinicians managing rare genetic disorders with multiple perplexing clinical manifestations.

Loss of function of SLC25A46 causes lethal congenital pontocerebellar hypoplasia.

Disturbed mitochondrial fusion and fission have been linked to various neurodegenerative disorders. In siblings from two unrelated families who died soon after birth with a profound neurodevelopmental disorder characterized by pontocerebellar hypoplasia and apnoea, we discovered a missense mutation and an exonic deletion in the SLC25A46 gene encoding a mitochondrial protein recently implicated in optic atrophy spectrum disorder. We performed functional studies that confirmed the mitochondrial localization and pro-fission properties of SLC25A46. Knockdown of slc24a46 expression in zebrafish embryos caused brain malformation, spinal motor neuron loss, and poor motility. At the cellular level, we observed abnormally elongated mitochondria, which was rescued by co-injection of the wild-type but not the mutant slc25a46 mRNA. Conversely, overexpression of the wild-type protein led to mitochondrial fragmentation and disruption of the mitochondrial network. In contrast to mutations causing non-lethal optic atrophy, missense mutations causing lethal congenital pontocerebellar hypoplasia markedly destabilize the protein. Indeed, the clinical severity appears inversely correlated with the relative stability of the mutant protein. This genotype-phenotype correlation underscores the importance of SLC25A46 and fine tuning of mitochondrial fission and fusion in pontocerebellar hypoplasia and central neurodevelopment in addition to optic and peripheral neuropathy across the life span.

Loss of ADAM17 is associated with severe multiorgan dysfunction

ADAM metallopeptidase domain 17 (ADAM17) is responsible for processing large numbers of proteins. Recently, 1 family involving 2 patients with a homozygous mutation in ADAM17 were described, presenting with skin lesions and diarrhea. In this report, we describe a second family confirming the existence of this syndrome. The proband presented with severe diarrhea, skin rash, and recurrent sepsis, eventually leading to her death at the age of 10 months. We performed exome sequencing and detailed pathological and immunological investigations. We identified a novel homozygous frameshift mutation in ADAM17 (NM_003183.4:c.308dupA) leading to a premature stop codon. CD4(+) and CD8(+) T-cell stimulation assays showed severely diminished tumor necrosis factor-α and interleukin-2 production. Skin biopsies indicated a focal neutrophilic infiltrate and spongiotic dermatitis. Interestingly, the patient developed unexplained systolic hypertension and nonspecific hepatitis with apoptosis. This report provides evidence for an important role of ADAM17 in human immunological response and underscores its multiorgan involvement.

A Novel Familial Mutation in the PCSK1 Gene That Alters the Oxyanion Hole Residue of Proprotein Convertase 1/3 and Impairs Its Enzymatic Activity

Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, encoding the neuroendocrine convertase 1 precursor (PC1/3) which was recently reported as a cause of Congenital Diarrhea Disorder (CDD). The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred.