Jeffrey G. Reid

Patterns and rates of exonic de novo mutations in autism spectrum disorders

Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified. To identify further genetic risk factors, here we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n = 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant, and the overall rate of mutation is only modestly higher than the expected rate. In contrast, the proteins encoded by genes that harboured de novo missense or nonsense mutations showed a higher degree of connectivity among themselves and to previous ASD genes as indexed by protein-protein interaction screens. The small increase in the rate of de novo events, when taken together with the protein interaction results, are consistent with an important but limited role for de novo point mutations in ASD, similar to that documented for de novo copy number variants. Genetic models incorporating these data indicate that most of the observed de novo events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant (that is, not necessarily sufficient for disease). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case–control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.

Clinical Whole-Exome Sequencing for the Diagnosis of Mendelian Disorders

BACKGROUND Whole-exome sequencing is a diagnostic approach for the identification of molecular defects in patients with suspected genetic disorders. METHODS We developed technical, bioinformatic, interpretive, and validation pipelines for whole-exome sequencing in a certified clinical laboratory to identify sequence variants underlying disease phenotypes in patients. RESULTS We present data on the first 250 probands for whom referring physicians ordered whole-exome sequencing. Patients presented with a range of phenotypes suggesting potential genetic causes. Approximately 80% were children with neurologic phenotypes. Insurance coverage was similar to that for established genetic tests. We identified 86 mutated alleles that were highly likely to be causative in 62 of the 250 patients, achieving a 25% molecular diagnostic rate (95% confidence interval, 20 to 31). Among the 62 patients, 33 had autosomal dominant disease, 16 had autosomal recessive disease, and 9 had X-linked disease. A total of 4 probands received two nonoverlapping molecular diagnoses, which potentially challenged the clinical diagnosis that had been made on the basis of history and physical examination. A total of 83% of the autosomal dominant mutant alleles and 40% of the X-linked mutant alleles occurred de novo. Recurrent clinical phenotypes occurred in patients with mutations that were highly likely to be causative in the same genes and in different genes responsible for genetically heterogeneous disorders. CONCLUSIONS Whole-exome sequencing identified the underlying genetic defect in 25% of consecutive patients referred for evaluation of a possible genetic condition. (Funded by the National Human Genome Research Institute.).

The Drosophila melanogaster Genetic Reference Panel

A major challenge of biology is understanding the relationship between molecular genetic variation and variation in quantitative traits, including fitness. This relationship determines our ability to predict phenotypes from genotypes and to understand how evolutionary forces shape variation within and between species. Previous efforts to dissect the genotype–phenotype map were based on incomplete genotypic information. Here, we describe the Drosophila melanogaster Genetic Reference Panel (DGRP), a community resource for analysis of population genomics and quantitative traits. The DGRP consists of fully sequenced inbred lines derived from a natural population. Population genomic analyses reveal reduced polymorphism in centromeric autosomal regions and the X chromosome, evidence for positive and negative selection, and rapid evolution of the X chromosome. Many variants in novel genes, most at low frequency, are associated with quantitative traits and explain a large fraction of the phenotypic variance. The DGRP facilitates genotype–phenotype mapping using the power of Drosophila genetics.

Whole-Genome Sequencing in a Patient with Charcot–Marie–Tooth Neuropathy

BACKGROUND Whole-genome sequencing may revolutionize medical diagnostics through rapid identification of alleles that cause disease. However, even in cases with simple patterns of inheritance and unambiguous diagnoses, the relationship between disease phenotypes and their corresponding genetic changes can be complicated. Comprehensive diagnostic assays must therefore identify all possible DNA changes in each haplotype and determine which are responsible for the underlying disorder. The high number of rare, heterogeneous mutations present in all humans and the paucity of known functional variants in more than 90% of annotated genes make this challenge particularly difficult. Thus, the identification of the molecular basis of a genetic disease by means of whole-genome sequencing has remained elusive. We therefore aimed to assess the usefulness of human whole-genome sequencing for genetic diagnosis in a patient with Charcot-Marie-Tooth disease. METHODS We identified a family with a recessive form of Charcot-Marie-Tooth disease for which the genetic basis had not been identified. We sequenced the whole genome of the proband, identified all potential functional variants in genes likely to be related to the disease, and genotyped these variants in the affected family members. RESULTS We identified and validated compound, heterozygous, causative alleles in SH3TC2 (the SH3 domain and tetratricopeptide repeats 2 gene), involving two mutations, in the proband and in family members affected by Charcot-Marie-Tooth disease. Separate subclinical phenotypes segregated independently with each of the two mutations; heterozygous mutations confer susceptibility to neuropathy, including the carpal tunnel syndrome. CONCLUSIONS As shown in this study of a family with Charcot-Marie-Tooth disease, whole-genome sequencing can identify clinically relevant variants and provide diagnostic information to inform the care of patients.

Molecular Findings Among Patients Referred for Clinical Whole-Exome Sequencing

IMPORTANCE Clinical whole-exome sequencing is increasingly used for diagnostic evaluation of patients with suspected genetic disorders. OBJECTIVE To perform clinical whole-exome sequencing and report (1) the rate of molecular diagnosis among phenotypic groups, (2) the spectrum of genetic alterations contributing to disease, and (3) the prevalence of medically actionable incidental findings such as FBN1 mutations causing Marfan syndrome. DESIGN, SETTING, AND PATIENTS Observational study of 2000 consecutive patients with clinical whole-exome sequencing analyzed between June 2012 and August 2014. Whole-exome sequencing tests were performed at a clinical genetics laboratory in the United States. Results were reported by clinical molecular geneticists certified by the American Board of Medical Genetics and Genomics. Tests were ordered by the patients physician. The patients were primarily pediatric (1756 [88%]; mean age, 6 years; 888 females [44%], 1101 males [55%], and 11 fetuses [1% gender unknown]), demonstrating diverse clinical manifestations most often including nervous system dysfunction such as developmental delay. MAIN OUTCOMES AND MEASURES Whole-exome sequencing diagnosis rate overall and by phenotypic category, mode of inheritance, spectrum of genetic events, and reporting of incidental findings. RESULTS A molecular diagnosis was reported for 504 patients (25.2%) with 58% of the diagnostic mutations not previously reported. Molecular diagnosis rates for each phenotypic category were 143/526 (27.2%; 95% CI, 23.5%-31.2%) for the neurological group, 282/1147 (24.6%; 95% CI, 22.1%-27.2%) for the neurological plus other organ systems group, 30/83 (36.1%; 95% CI, 26.1%-47.5%) for the specific neurological group, and 49/244 (20.1%; 95% CI, 15.6%-25.8%) for the nonneurological group. The Mendelian disease patterns of the 527 molecular diagnoses included 280 (53.1%) autosomal dominant, 181 (34.3%) autosomal recessive (including 5 with uniparental disomy), 65 (12.3%) X-linked, and 1 (0.2%) mitochondrial. Of 504 patients with a molecular diagnosis, 23 (4.6%) had blended phenotypes resulting from 2 single gene defects. About 30% of the positive cases harbored mutations in disease genes reported since 2011. There were 95 medically actionable incidental findings in genes unrelated to the phenotype but with immediate implications for management in 92 patients (4.6%), including 59 patients (3%) with mutations in genes recommended for reporting by the American College of Medical Genetics and Genomics. CONCLUSIONS AND RELEVANCE Whole-exome sequencing provided a potential molecular diagnosis for 25% of a large cohort of patients referred for evaluation of suspected genetic conditions, including detection of rare genetic events and new mutations contributing to disease. The yield of whole-exome sequencing may offer advantages over traditional molecular diagnostic approaches in certain patients.

Complete Khoisan and Bantu genomes from southern Africa

The genetic structure of the indigenous hunter-gatherer peoples of southern Africa, the oldest known lineage of modern human, is important for understanding human diversity. Studies based on mitochondrial and small sets of nuclear markers have shown that these hunter-gatherers, known as Khoisan, San, or Bushmen, are genetically divergent from other humans. However, until now, fully sequenced human genomes have been limited to recently diverged populations. Here we present the complete genome sequences of an indigenous hunter-gatherer from the Kalahari Desert and a Bantu from southern Africa, as well as protein-coding regions from an additional three hunter-gatherers from disparate regions of the Kalahari. We characterize the extent of whole-genome and exome diversity among the five men, reporting 1.3 million novel DNA differences genome-wide, including 13,146 novel amino acid variants. In terms of nucleotide substitutions, the Bushmen seem to be, on average, more different from each other than, for example, a European and an Asian. Observed genomic differences between the hunter-gatherers and others may help to pinpoint genetic adaptations to an agricultural lifestyle. Adding the described variants to current databases will facilitate inclusion of southern Africans in medical research efforts, particularly when family and medical histories can be correlated with genome-wide data.

Mind the Gap: Upgrading Genomes with Pacific Biosciences RS Long-Read Sequencing Technology

Many genomes have been sequenced to high-quality draft status using Sanger capillary electrophoresis and/or newer short-read sequence data and whole genome assembly techniques. However, even the best draft genomes contain gaps and other imperfections due to limitations in the input data and the techniques used to build draft assemblies. Sequencing biases, repetitive genomic features, genomic polymorphism, and other complicating factors all come together to make some regions difficult or impossible to assemble. Traditionally, draft genomes were upgraded to “phase 3 finished” status using time-consuming and expensive Sanger-based manual finishing processes. For more facile assembly and automated finishing of draft genomes, we present here an automated approach to finishing using long-reads from the Pacific Biosciences RS (PacBio) platform. Our algorithm and associated software tool, PBJelly, (publicly available at https://sourceforge.net/projects/pb-jelly/) automates the finishing process using long sequence reads in a reference-guided assembly process. PBJelly also provides “lift-over” co-ordinate tables to easily port existing annotations to the upgraded assembly. Using PBJelly and long PacBio reads, we upgraded the draft genome sequences of a simulated Drosophila melanogaster, the version 2 draft Drosophila pseudoobscura, an assembly of the Assemblathon 2.0 budgerigar dataset, and a preliminary assembly of the Sooty mangabey. With 24× mapped coverage of PacBio long-reads, we addressed 99% of gaps and were able to close 69% and improve 12% of all gaps in D. pseudoobscura. With 4× mapped coverage of PacBio long-reads we saw reads address 63% of gaps in our budgerigar assembly, of which 32% were closed and 63% improved. With 6.8× mapped coverage of mangabey PacBio long-reads we addressed 97% of gaps and closed 66% of addressed gaps and improved 19%. The accuracy of gap closure was validated by comparison to Sanger sequencing on gaps from the original D. pseudoobscura draft assembly and shown to be dependent on initial reference quality.

Whole-Genome Sequencing for Optimized Patient Management

A disease mutation identified by whole-genome sequencing of twins with dystonia allowed optimization of treatment, resulting in clinical improvements. Guiding Treatment with Genomics Whole-genome sequencing of DNA from patients with different diseases is proving useful for identifying new disease-causing mutations, but can it help physicians make better decisions about treatment options for these patients? A new study by Bainbridge and colleagues suggests that it can. Bainbridge et al. sequenced the complete genomes of a male and female fraternal twin pair, who had been diagnosed 9 years earlier with the movement disorder dopa (3,4-dihydroxyphenylalanine)–responsive dystonia (DRD). This complex disorder is difficult to diagnose and may be mistaken for other movement disorders involving loss of the neurotransmitter dopamine. The standard treatment for DRD is to replace dopamine by providing a dopamine precursor called l-dopa, the drug that is also used to treat the common movement disorder Parkinson disease. When the twins were diagnosed with DRD, they seemed to fit the classic description of DRD and were given l-dopa, which did help to alleviate many of their symptoms. When Bainbridge and colleagues analyzed the full genome sequences of the twins, they were surprised to discover no mutations in the two genes most commonly mutated in DRD. Instead, they pinpointed a mutation in the SPR gene encoding sepiapterin reductase, which synthesizes a cofactor needed for the action of enzymes that make not only dopamine but also the neurotransmitter serotonin. This finding suggested to the authors that supplementing the twin’s current l-dopa treatment with a serotonin precursor, 5-hydroxytryptophan, might provide further improvement in their symptoms. Sure enough, when the twins were given both l-dopa and 5-hydroxytryptophan instead of l-dopa alone, they showed improvement in their symptoms after 1 to 2 weeks, including greater attention in school, better motion and coordination, and reduced hand tremor as evidenced by more legible handwriting. Although this study involved only one twin pair, it does demonstrate how whole-genome sequencing could be applied to glean more detailed information about a patient’s disease, leading to more optimized treatment and a better outcome. Whole-genome sequencing of patient DNA can facilitate diagnosis of a disease, but its potential for guiding treatment has been under-realized. We interrogated the complete genome sequences of a 14-year-old fraternal twin pair diagnosed with dopa (3,4-dihydroxyphenylalanine)–responsive dystonia (DRD; Mendelian Inheritance in Man #128230). DRD is a genetically heterogeneous and clinically complex movement disorder that is usually treated with l-dopa, a precursor of the neurotransmitter dopamine. Whole-genome sequencing identified compound heterozygous mutations in the SPR gene encoding sepiapterin reductase. Disruption of SPR causes a decrease in tetrahydrobiopterin, a cofactor required for the hydroxylase enzymes that synthesize the neurotransmitters dopamine and serotonin. Supplementation of l-dopa therapy with 5-hydroxytryptophan, a serotonin precursor, resulted in clinical improvements in both twins.

Expression profiling of microRNAs by deep sequencing

MicroRNAs are short non-coding RNAs that regulate the stability and translation of mRNAs. Profiling experiments, using microarray or deep sequencing technology, have identified microRNAs that are preferentially expressed in certain tissues, specific stages of development, or disease states such as cancer. Deep sequencing utilizes massively parallel sequencing, generating millions of small RNA sequence reads from a given sample. Profiling of microRNAs by deep sequencing measures absolute abundance and allows for the discovery of novel microRNAs that have eluded previous cloning and standard sequencing efforts. Public databases provide in silico predictions of microRNA gene targets by various algorithms. To better determine which of these predictions represent true positives, microRNA expression data can be integrated with gene expression data to identify putative microRNA:mRNA functional pairs. Here we discuss tools and methodologies for the analysis of microRNA expression data from deep sequencing.

A Link between mir-100 and FRAP1/mTOR in Clear Cell Ovarian Cancer

MicroRNAs (miRNAs) are small noncoding RNAs that direct gene regulation through translational repression and degradation of complementary mRNA. Although miRNAs have been implicated as oncogenes and tumor suppressors in a variety of human cancers, functional roles for individual miRNAs have not been described in clear cell ovarian carcinoma, an aggressive and chemoresistant subtype of ovarian cancer. We performed deep sequencing to comprehensively profile miRNA expression in 10 human clear cell ovarian cancer cell lines compared with normal ovarian surface epithelial cultures and discovered 54 miRNAs that were aberrantly expressed. Because of the critical roles of the phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene homolog 1/mammalian target of rapamycin (mTOR) pathway in clear cell ovarian cancer, we focused on mir-100, a putative tumor suppressor that was the most down-regulated miRNA in our cancer cell lines, and its up-regulated target, FRAP1/mTOR. Overexpression of mir-100 inhibited mTOR signaling and enhanced sensitivity to the rapamycin analog RAD001 (everolimus), confirming the key relationship between mir-100 and the mTOR pathway. Furthermore, overexpression of the putative tumor suppressor mir-22 repressed the EVI1 oncogene, which is known to suppress apoptosis by stimulating phosphatidylinositol 3-kinase/v-akt murine thymoma viral oncogene homolog 1 signaling. In addition to these specific effects, reversing the expression of mir-22 and the putative oncogene mir-182 had widespread effects on target and nontarget gene populations that ultimately caused a global shift in the cancer gene signature toward a more normal state. Our experiments have revealed strong candidate miRNAs and their target genes that may contribute to the pathogenesis of clear cell ovarian cancer, thereby highlighting alternative therapeutic strategies for the treatment of this deadly cancer.