Claudia Gonzaga-Jauregui

Integrating common and rare genetic variation in diverse human populations.

Despite great progress in identifying genetic variants that influence human disease, most inherited risk remains unexplained. A more complete understanding requires genome-wide studies that fully examine less common alleles in populations with a wide range of ancestry. To inform the design and interpretation of such studies, we genotyped 1.6 million common single nucleotide polymorphisms (SNPs) in 1,184 reference individuals from 11 global populations, and sequenced ten 100-kilobase regions in 692 of these individuals. This integrated data set of common and rare alleles, called ‘HapMap 3’, includes both SNPs and copy number polymorphisms (CNPs). We characterized population-specific differences among low-frequency variants, measured the improvement in imputation accuracy afforded by the larger reference panel, especially in imputing SNPs with a minor allele frequency of ≤5%, and demonstrated the feasibility of imputing newly discovered CNPs and SNPs. This expanded public resource of genome variants in global populations supports deeper interrogation of genomic variation and its role in human disease, and serves as a step towards a high-resolution map of the landscape of human genetic variation.

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.

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.

Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome.

We identified complex genomic rearrangements consisting of intermixed duplications and triplications of genomic segments at the MECP2 and PLP1 loci. These complex rearrangements were characterized by a triplicated segment embedded within a duplication in 11 unrelated subjects. Notably, only two breakpoint junctions were generated during each rearrangement formation. All the complex rearrangement products share a common genomic organization, duplication-inverted triplication-duplication (DUP-TRP/INV-DUP), in which the triplicated segment is inverted and located between directly oriented duplicated genomic segments. We provide evidence that the DUP-TRP/INV-DUP structures are mediated by inverted repeats that can be separated by >300 kb, a genomic architecture that apparently leads to susceptibility to such complex rearrangements. A similar inverted repeat–mediated mechanism may underlie structural variation in many other regions of the human genome. We propose a mechanism that involves both homology-driven events, via inverted repeats, and microhomologous or nonhomologous events.

Human CLP1 Mutations Alter tRNA Biogenesis, Affecting Both Peripheral and Central Nervous System Function

CLP1 is a RNA kinase involved in tRNA splicing. Recently, CLP1 kinase-dead mice were shown to display a neuromuscular disorder with loss of motor neurons and muscle paralysis. Human genome analyses now identified a CLP1 homozygous missense mutation (p.R140H) in five unrelated families, leading to a loss of CLP1 interaction with the tRNA splicing endonuclease (TSEN) complex, largely reduced pre-tRNA cleavage activity, and accumulation of linear tRNA introns. The affected individuals develop severe motor-sensory defects, cortical dysgenesis, and microcephaly. Mice carrying kinase-dead CLP1 also displayed microcephaly and reduced cortical brain volume due to the enhanced cell death of neuronal progenitors that is associated with reduced numbers of cortical neurons. Our data elucidate a neurological syndrome defined by CLP1 mutations that impair tRNA splicing. Reduction of a founder mutation to homozygosity illustrates the importance of rare variations in disease and supports the clan genomics hypothesis.

Distribution and clinical impact of functional variants in 50,726 whole-exome sequences from the DiscovEHR study

Unleashing the power of precision medicine Precision medicine promises the ability to identify risks and treat patients on the basis of pathogenic genetic variation. Two studies combined exome sequencing results for over 50,000 people with their electronic health records. Dewey et al. found that ∼3.5% of individuals in their cohort had clinically actionable genetic variants. Many of these variants affected blood lipid levels that could influence cardiovascular health. Abul-Husn et al. extended these findings to investigate the genetics and treatment of familial hypercholesterolemia, a risk factor for cardiovascular disease, within their patient pool. Genetic screening helped identify at-risk patients who could benefit from increased treatment. Science, this issue p. 10.1126/science.aaf6814, p. 10.1126/science.aaf7000 More than 50,000 exomes, coupled with electronic health records, inform on medically relevant genetic variants. INTRODUCTION Large-scale genetic studies of integrated health care populations, with phenotypic data captured natively in the documentation of clinical care, have the potential to unveil genetic associations that point the way to new biology and therapeutic targets. This setting also represents an ideal test bed for the implementation of genomics in routine clinical care in service of precision medicine. RATIONALE The DiscovEHR collaboration between the Regeneron Genetics Center and Geisinger Health System aims to catalyze genomic discovery and precision medicine by coupling high-throughput exome sequencing to longitudinal electronic health records (EHRs) of participants in Geisinger’s MyCode Community Health Initiative. Here, we describe initial insights from whole-exome sequencing of 50,726 adult participants of predominantly European ancestry using clinical phenotypes derived from EHRs. RESULTS The median duration of EHR data associated with sequenced participants was 14 years, with a median of 87 clinical encounters, 687 laboratory tests, and seven procedures per participant. Forty-eight percent of sequenced individuals had one or more first- or second-degree relatives in the sample, and genome-wide autozygosity was similar to other outbred European populations. We found ~4.2 million single-nucleotide variants and insertion/deletion events, of which ~176,000 are predicted to result in loss of gene function (LoF). The overwhelming majority of these genetic variants occurred at a minor allele frequency of ≤1%, and more than half were singletons. Each participant harbored a median of 21 rare predicted LoFs. At this sample size, ~92% of sequenced genes, including genes that encode existing drug targets or confer risk for highly penetrant genetic diseases, harbor rare heterozygous predicted LoF variants. About 7% of sequenced genes contained rare homozygous predicted LoF variants in at least one individual. Linking these data to EHR-derived laboratory phenotypes revealed consequences of partial or complete LoF in humans. Among these were previously unidentified associations between predicted LoFs in CSF2RB and basophil and eosinophil counts, and EGLN1-associated erythrocytosis segregating in genetically identified family networks. Using predicted LoFs as a model for drug target antagonism, we found associations supporting the majority of therapeutic targets for lipid lowering. To highlight the opportunity for genotype-phenotype association discovery, we performed exome-wide association analyses of EHR-derived lipid values, newly implicating rare predicted LoFs, and deleterious missense variants in G6PC in association with triglyceride levels. In a survey of 76 clinically actionable disease-associated genes, we estimated that 3.5% of individuals harbor pathogenic or likely pathogenic variants that meet criteria for clinical action. Review of the EHR uncovered findings associated with the monogenic condition in ~65% of pathogenic variant carriers’ medical records. CONCLUSION The findings reported here demonstrate the value of large-scale sequencing in an integrated health system population, add to the knowledge base regarding the phenotypic consequences of human genetic variation, and illustrate the challenges and promise of genomic medicine implementation. DiscovEHR provides a blueprint for large-scale precision medicine initiatives and genomics-guided therapeutic target discovery. Therapeutic target validation and genomic medicine in DiscovEHR. (A) Associations between predicted LoF variants in lipid drug target genes and lipid levels. Boxes correspond to effect size, given as the absolute value of effect, in SD units; whiskers denote 95% confidence intervals for effect. The size of the box is proportional to the logarithm (base 10) of predicted LoF carriers. (B and C) Prevalence and expressivity of clinically actionable genetic variants in 76 disease genes, according to EHR data. G76, Geisinger-76. The DiscovEHR collaboration between the Regeneron Genetics Center and Geisinger Health System couples high-throughput sequencing to an integrated health care system using longitudinal electronic health records (EHRs). We sequenced the exomes of 50,726 adult participants in the DiscovEHR study to identify ~4.2 million rare single-nucleotide variants and insertion/deletion events, of which ~176,000 are predicted to result in a loss of gene function. Linking these data to EHR-derived clinical phenotypes, we find clinical associations supporting therapeutic targets, including genes encoding drug targets for lipid lowering, and identify previously unidentified rare alleles associated with lipid levels and other blood level traits. About 3.5% of individuals harbor deleterious variants in 76 clinically actionable genes. The DiscovEHR data set provides a blueprint for large-scale precision medicine initiatives and genomics-guided therapeutic discovery.

Exome Sequence Analysis Suggests that Genetic Burden Contributes to Phenotypic Variability and Complex Neuropathy

Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous distal symmetric polyneuropathy. Whole-exome sequencing (WES) of 40 individuals from 37 unrelated families with CMT-like peripheral neuropathy refractory to molecular diagnosis identified apparent causal mutations in ∼ 45% (17/37) of families. Three candidate disease genes are proposed, supported by a combination of genetic and in vivo studies. Aggregate analysis of mutation data revealed a significantly increased number of rare variants across 58 neuropathy-associated genes in subjects versus controls, confirmed in a second ethnically discrete neuropathy cohort, suggesting that mutation burden potentially contributes to phenotypic variability. Neuropathy genes shown to have highly penetrant Mendelizing variants (HPMVs) and implicated by burden in families were shown to interact genetically in a zebrafish assay exacerbating the phenotype established by the suppression of single genes. Our findings suggest that the combinatorial effect of rare variants contributes to disease burden and variable expressivity.

Genetic identification of familial hypercholesterolemia within a single U.S. health care system

Unleashing the power of precision medicine Precision medicine promises the ability to identify risks and treat patients on the basis of pathogenic genetic variation. Two studies combined exome sequencing results for over 50,000 people with their electronic health records. Dewey et al. found that ∼3.5% of individuals in their cohort had clinically actionable genetic variants. Many of these variants affected blood lipid levels that could influence cardiovascular health. Abul-Husn et al. extended these findings to investigate the genetics and treatment of familial hypercholesterolemia, a risk factor for cardiovascular disease, within their patient pool. Genetic screening helped identify at-risk patients who could benefit from increased treatment. Science, this issue p. 10.1126/science.aaf6814, p. 10.1126/science.aaf7000 Genomic screening can prompt the diagnosis of familial hypercholesterolemia patients, the majority of whom are receiving inadequate lipid-lowering therapy. INTRODUCTION Familial hypercholesterolemia (FH) is a public health genomics priority but remains underdiagnosed and undertreated despite widespread cholesterol screening. This represents a missed opportunity to prevent FH-associated cardiovascular morbidity and mortality. Pathogenic variants in three genes (LDLR, APOB, and PCSK9) account for the majority of FH cases. We assessed the prevalence and clinical impact of FH-associated genomic variants in 50,726 individuals from the MyCode Community Health Initiative at Geisinger Health System who underwent exome sequencing as part of the DiscovEHR human genetics collaboration with the Regeneron Genetics Center. RATIONALE Genetic testing for FH is uncommon in clinical practice in the United States, and the prevalence of FH variants in U.S. populations has not been well established. We sought to evaluate FH prevalence in a large integrated U.S. health care system using genomic sequencing and electronic health record (EHR) data. We determined the impact of FH variants on low-density lipoprotein cholesterol (LDL-C) levels and coronary artery disease (CAD) risk. We assessed the likelihood of FH variant carriers achieving a presequencing EHR-based FH diagnosis according to established clinical diagnostic criteria. Finally, we examined the rates of statin medication use and outcomes in FH variant carriers. RESULTS Thirty-five known and predicted pathogenic variants in LDLR, APOB, and PCSK9 were identified in 229 individuals. The estimated FH prevalence was 1:256 in unselected participants and 1:118 in participants ascertained via the cardiac catheterization laboratory. FH variants were found in only 2.5% of individuals with severe hypercholesterolemia (maximum EHR-documented LDL-C ≥ 190 mg/dl) in the cohort, and a maximum LDL-C of ≥190 mg/dl was absent in 45% of FH variant carriers. Overall, FH variant carriers had 69 ± 3 mg/dl greater maximum LDL-C than sequenced noncarriers (P = 1.8 × 10−20) and had significantly increased odds of general and premature CAD [odds ratio (OR), 2.6 (P = 4.3 × 10−11) and 3.7 (P = 5.5 × 10−14), respectively]. The increased odds of general and premature CAD were most pronounced in carriers of LDLR predicted loss-of-function variants [OR, 5.5 (P = 7.7 × 10−13) and 10.3 (P = 9.8 × 10−19), respectively]. Fourteen FH variant carriers were deceased; chart review revealed that none of these individuals had a clinical diagnosis of FH. Before genetic testing, only 15% of FH variant carriers had an ICD-10 (International Classification of Diseases, 10th revision) diagnosis code for pure hypercholesterolemia or had been seen in a lipid clinic, suggesting that few had been previously diagnosed with FH. Retrospectively applying Dutch Lipid Clinic Network diagnostic criteria to EHR data, we found presequencing criteria supporting a probable or definite clinical diagnosis of FH in 24% of FH variant carriers, highlighting the limitations of using existing clinical criteria for EHR-based screening in the absence of genetic testing. Active statin use was identified in 58% and high-intensity statin use in 37% of FH variant carriers. Only 46% of carriers currently on statin therapy had a most recent LDL-C level below 100 mg/dl compared to 77% of noncarriers. CONCLUSION In summary, we show that large-scale genomic screening in patients with longitudinal EHR data has the ability to detect FH, uncover and characterize novel pathogenic variants, determine disease prevalence, and enhance overall knowledge of clinical impact and outcomes. The 1:256 prevalence of FH variants in this predominantly European-American cohort is in line with prevalence estimates from recent work in European cohorts. Our findings highlight the undertreatment of FH variant carriers and demonstrate a potential clinical benefit for large-scale sequencing initiatives in service of precision medicine. Prevalence and clinical impact of FH variants in a large U.S. clinical care cohort. (A) Distribution of 229 heterozygous carriers of an FH variant in the DiscovEHR cohort by FH gene. (B) Prevalence of an FH variant in the DiscovEHR cohort and according to recruitment site

Heterozygous De Novo and Inherited Mutations in the Smooth Muscle Actin (ACTG2) Gene Underlie Megacystis-Microcolon-Intestinal Hypoperistalsis Syndrome

Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS) is a rare disorder of enteric smooth muscle function affecting the intestine and bladder. Patients with this severe phenotype are dependent on total parenteral nutrition and urinary catheterization. The cause of this syndrome has remained a mystery since Berdons initial description in 1976. No genes have been clearly linked to MMIHS. We used whole-exome sequencing for gene discovery followed by targeted Sanger sequencing in a cohort of patients with MMIHS and intestinal pseudo-obstruction. We identified heterozygous ACTG2 missense variants in 15 unrelated subjects, ten being apparent de novo mutations. Ten unique variants were detected, of which six affected CpG dinucleotides and resulted in missense mutations at arginine residues, perhaps related to biased usage of CpG containing codons within actin genes. We also found some of the same heterozygous mutations that we observed as apparent de novo mutations in MMIHS segregating in families with intestinal pseudo-obstruction, suggesting that ACTG2 is responsible for a spectrum of smooth muscle disease. ACTG2 encodes γ2 enteric actin and is the first gene to be clearly associated with MMIHS, suggesting an important role for contractile proteins in enteric smooth muscle disease.

COPA mutations impair ER-Golgi transport and cause hereditary autoimmune-mediated lung disease and arthritis

Unbiased genetic studies have uncovered surprising molecular mechanisms in human cellular immunity and autoimmunity. We performed whole-exome sequencing and targeted sequencing in five families with an apparent mendelian syndrome of autoimmunity characterized by high-titer autoantibodies, inflammatory arthritis and interstitial lung disease. We identified four unique deleterious variants in the COPA gene (encoding coatomer subunit α) affecting the same functional domain. Hypothesizing that mutant COPA leads to defective intracellular transport via coat protein complex I (COPI), we show that COPA variants impair binding to proteins targeted for retrograde Golgi-to-ER transport. Additionally, expression of mutant COPA results in ER stress and the upregulation of cytokines priming for a T helper type 17 (TH17) response. Patient-derived CD4+ T cells also demonstrate significant skewing toward a TH17 phenotype that is implicated in autoimmunity. Our findings uncover an unexpected molecular link between a vesicular transport protein and a syndrome of autoimmunity manifested by lung and joint disease.