Whole Genome Sequencing In Patients Research Articles

Longitudinal cell-free DNA characterization by low-coverage whole-genome sequencing in patients undergoing high-dose radiotherapy

Background and PurposeCurrent radiotherapy guidelines rely heavily on imaging-based monitoring. Liquid biopsy monitoring promises to complement imaging by providing frequent systemic information about the tumor. In particular, cell-free DNA (cfDNA) sequencing offers a tumor-agnostic approach, which lends itself to monitoring heterogeneous cohorts of cancer patients. MethodsWe collected plasma cfDNA from oligometastatic patients (OMD) and head-and-neck cancer patients (SCCHN) at six time points before, during, and after radiotherapy, and compared them to the plasma samples of healthy and polymetastatic volunteers. We performed low-pass (on average 7x) whole-genome sequencing on 93 plasma cfDNA samples and correlated copy number alterations and fragment length distributions to clinical and imaging findings. ResultsWe observed copy number alterations in 4/7 polymetastatic cancer patients, 1/7 OMD and 1/7 SCCHN patients, these patients’ imaging showed progression following radiotherapy. Using unsupervised learning, we identified cancer-specific fragment length features that showed a strong correlation with copy number-based tumor fraction estimates. In 4/4 HPV-positive SCCHN patient samples, we detected viral DNA that enabled the monitoring of very low tumor fraction samples. ConclusionsOur results indicate that an elevated tumor fraction is associated with tumor aggressiveness and systemic tumor spread. This information may be used to adapt treatment strategies. Further, we show that by detecting specific sequences such as viral DNA, the sensitivity of detecting cancer from cell-free DNA sequencing data can be greatly increased.

Whole-genome sequencing in clinically diagnosed Charcot-Marie-Tooth disease undiagnosed by whole-exome sequencing.

Whole-genome sequencing is the most comprehensive form of next-generation sequencing method. We aimed to assess the additional diagnostic yield of whole-genome sequencing in patients with clinically diagnosed Charcot-Marie-Tooth disease when compared with whole-exome sequencing, which has not been reported in the literature. Whole-genome sequencing was performed on 72 families whose genetic cause of clinically diagnosed Charcot-Marie-Tooth disease was not revealed after the whole-exome sequencing and 17p12 duplication screening. Among the included families, 14 (19.4%) acquired genetic diagnoses that were compatible with their phenotypes. The most common factor that led to the additional diagnosis in the whole-genome sequencing was genotype-driven analysis (four families, 4/14), in which a wider range of genes, not limited to peripheral neuropathy-related genes, were analysed. Another four families acquired diagnosis due to the inherent advantage of whole-genome sequencing such as better coverage than the whole-exome sequencing (two families, 2/14), structural variants (one family, 1/14) and non-coding variants (one family, 1/14). In conclusion, an evident gain in diagnostic yield was obtained from whole-genome sequencing of the whole-exome sequencing-negative cases. A wide range of genes, not limited to inherited peripheral neuropathy-related genes, should be targeted during whole-genome sequencing.

CT Radiomics and Whole Genome Sequencing in Patients with Pancreatic Ductal Adenocarcinoma: Predictive Radiogenomics Modeling.

We investigate whether computed tomography (CT) derived radiomics may correlate with driver gene mutations in patients with pancreatic ductal adenocarcinoma (PDAC). In this retrospective study, 47 patients (mean age 64 ± 11 years; range: 42-86 years) with PDAC, who were treated surgically and who underwent preoperative CT imaging at our institution were included in the study. Image segmentation and feature extraction was performed semi-automatically with a commonly used open-source software platform. Genomic data from whole genome sequencing (WGS) were collected from our institution's web-based resource. Two statistical models were then built, in order to evaluate the predictive ability of CT-derived radiomics feature for driver gene mutations in PDAC. 30/47 of all tumor samples harbored 2 or more gene mutations. Overall, 81% of tumor samples demonstrated mutations in KRAS, 68% of samples had alterations in TP53, 26% in SMAD4 and 19% in CDKN2A. Extended statistical analysis revealed acceptable predictive ability for KRAS and TP53 (Youden Index 0.56 and 0.67, respectively) and mild to acceptable predictive signal for SMAD4 and CDKN2A (Youden Index 0.5, respectively). Our study establishes acceptable correlation of radiomics features and driver gene mutations in PDAC, indicating an acceptable prognostication of genomic profiles using CT-derived radiomics. A larger and more homogenous cohort may further enhance the predictive ability.

Clinical impact of whole-genome sequencing in patients with early-onset dementia

BackgroundIn the clinical setting, identification of the genetic cause in patients with early-onset dementia (EOD) is challenging due to multiple types of genetic tests required to arrive at a diagnosis....

DOCKopathies: A systematic review of the clinical pathologies associated with human DOCK pathogenic variants.

The Dedicator of Cytokinesis (DOCK) family (DOCK1-11) of genes are essential mediators of cellular migration, growth, and fusion in a variety of cell types and tissues. Recent advances in whole-genome sequencingof patients with undiagnosed genetic disorders have identified several rare pathogenic variants in DOCK genes. We conducted a systematic review and performed a patient database and literature search of reported DOCK pathogenic variants that have been identified in association with clinical pathologies such as global developmental delay, immune cell dysfunction, muscle hypotonia, and muscle ataxia among other categories. We then categorized these pathogenic DOCK variants and their associated clinical phenotypes under several unique categories: developmental, cardiovascular, metabolic, cognitive, or neuromuscular. Our systematic review of DOCK variants aims to identify and analyze potential DOCK-regulated networks associated with neuromuscular diseases and other disease pathologies, which may identify novel therapeutic strategies and targets. This systematic analysis and categorization of human-associated pathologies with DOCK pathogenic variants is the first report to the best of our knowledge for a unique class in this understudied gene family that has important implications in furthering personalized genomic medicine, clinical diagnoses, and improve targeted therapeutic outcomes across many clinical pathologies.

Complementary Experimental Methods in Genetics Open Up New Avenues of Research to Elucidate the Pathogenesis of Periodontitis.

A complex disease such as periodontitis is the sum of environmental and genetic effects. The personal genetic constitution interacts with the effects of internal and external risk factors like smoking, oral hygiene, malnutrition, emotional stress, and age. Accordingly, individuals who live in the same environmental context and share comparable lifestyle habits have different disease risks. Genetic research offers the identification of DNA sequence variants that have a causal role in disease etiology and allows the identification of disease relevant immune and metabolic pathways that contribute to disease susceptibility and pathogenesis in specific situations. Real advances have been made in genetic medical research in the last years. Starting from candidate gene association studies, new approaches were employed that have expanded the study design of genomewide association studies to genomewide meta-analyses and gene x environment interaction studies. Cost efficient whole-exome and whole-genome sequencing of patients with rare severe forms of periodontitis has the potential to identify genes and pathways with a direct role in the pathogenesis of common forms. In parallel, animal models were developed that use genetically highly diverse mouse lines to identify risk genes of human diseases. This chapter presents the main studies and the identified susceptibility genes that have clear statistical evidence. In addition, it describes pioneering studies that used advanced methods in experimental dental research, opening up new avenues of research. Although the knowledge of the genetic architecture of periodontitis is still in its infancy, genetic research is building the basis for future works with the potential to advance dental medicine in ways that will determine the various causes of periodontal diseases. This knowledge may eventually allow making predictions about disease risk for individual patients and leading to diagnosis and treatments that do not treat the symptoms but heal the disease.

Whole-genome sequencing of patients with rare diseases in a national health system

The authors applied whole-genome sequencing (WGS) in 9,802 patients with a rare disease in a national health system to streamline diagnosis and to discover unknown aetiological variants in the coding and non-coding regions of the genome. WGS identified the genetic diagnosis in 1138/7065 extensively phenotyped participants. They identified 95 genes in which mutations were very likely to be the cause of a rare Mendelian disease.

Complete characterisation of two new large Xq28 duplications involving F8 using whole genome sequencing in patients without haemophilia A.

Depending on the location of insertion of the gained region, F8 duplications can have variable clinical impacts from benign impact to severe haemophilia A phenotype. To characterize two large Xq28 duplications involving F8 incidentally detected by chromosome microarray analysis (CMA) in two patients presenting severe intellectual disability but no history of bleeding disorder. Whole genome sequencing (WGS) was performed in order to characterize the two large Xq28 duplications at nucleotide level. In patient 1, a 60-73kb gained region encompassing the exons 23-26 of F8 and SMIM9 was inserted at the int22h-2 locus following a non-homologous recombination between int22h-1 and int22h-2. We hypothesized that two independent events, micro-homology-mediated break-induced replication (MMBIR) and break-induced replication (BIR), could be involved in this rearrangement. In patient 2, the CMA found duplication from 101 to 116-kb long encompassing the exons 16-26 of F8 and SMIM9. The WGS analysis identified a more complex rearrangement with the presence of three genomic junctions. Due to the multiple micro-homologies observed at breakpoints, a replication-based mechanism such as fork stalling and template switching (FoSTeS) was greatly suspected. In both cases, these complex rearrangements preserved an intact copy of the F8. This study highlights the value of WGS to characterize the genomic junction at the nucleotide level and ultimately better describe the molecular mechanisms involved in Xq28 structural variations. It also emphasizes the importance of specifying the structure of the genomic gain in order to improve genotype-phenotype correlation and genetic counselling.

Novel Compound Heterozygous Mutation in TRAPPC9 Gene: The Relevance of Whole Genome Sequencing

Advances in high-throughput technologies and its implementation worldwide have had a considerable impact on the elucidation of the molecular causes underlying neurodevelopmental psychiatric disorders, especially for autism spectrum disorder and intellectual disability (ID). Nevertheless, etiology remains elusive in close to 50% of cases, even in those families with multiple affected individuals, strongly hinting at a genetic cause. Here we present a case report of two siblings affected with severe ID and other comorbidities, who embarked on a genetic testing odyssey until diagnosis was reached by using whole genome sequencing (WGS). WGS identified a maternally inherited novel missense variant (NM_031466.7:c.1037G > A; p.Gly346Glu) and a paternally inherited 90 kb intragenic deletion in TRAPPC9 gene. This report demonstrates the clinical utility of WGS in patients who remain undiagnosed after whole exome sequencing.

Whole-genome sequencing of patients with rare diseases in a national health system.

Most patients with rare diseases do not receive a molecular diagnosis and the aetiological variants and causative genes for more than half such disorders remain to be discovered1. Here we used whole-genome sequencing (WGS) in a national health system to streamline diagnosis and to discover unknown aetiological variants in the coding and non-coding regions of the genome. We generated WGS data for 13,037 participants, of whom 9,802 had a rare disease, and provided a genetic diagnosis to 1,138 of the 7,065extensively phenotypedparticipants. We identified 95 Mendelian associations between genes and rare diseases, of which 11 have been discovered since 2015 and at least 79 are confirmed to be aetiological. By generating WGS data ofUK Biobankparticipants2, we found that rare alleles can explain the presence of some individuals in the tails of a quantitative trait for red blood cells. Finally, we identified four novel non-coding variants that cause disease through the disruption of transcription of ARPC1B, GATA1, LRBA and MPL. Our study demonstrates a synergy by using WGS for diagnosis and aetiological discovery in routine healthcare.

P.295Linked-read whole genome sequencing in patients with congenital myopathy

Over the past decade, Next Generation Sequencing (NGS) methods have revolutionised the field of genetics. Currently, the usage of Whole Genome Sequencing (WGS) in addition to Whole Exome Sequencing (WES) and panel sequencing is increasing in research and in clinical diagnostics. In this study, we wanted to test whether a new NGS application — the Linked-Read (LR) library preparation — can improve the identification of disease-causing variants in patients with congenital myopathy. For LR library preparation, gDNA molecules are encapsulated inside emulsion droplets. Every microreaction provides a unique barcode to each of the DNA fragments inside the droplet. In the data analysis step, the barcoded short-read sequences are linked up to each other, providing long-range information. In addition to regular WGS information, this enables the identification of structural variants such as insertions or translocations. It also resolves the haplotypes of the patient. Moreover, LRs have the potential to improve the short-read alignment of repetitive regions in genes such as nebulin and titin. We selected three patients with a congenital myopathy for this pilot study. The prior analysis using targeted Sanger sequencing of candidate genes, a custom neuromuscular gene panel or WES had failed to identify the genetic cause of their disorder. Furthermore, all patient samples had previously been analysed for Copy Number Variation (CNV) using a Comparative Genomic Hybridisation array (CGH-array) for neuromuscular disorders designed by our group, but the molecular diagnosis had remained unresolved. Our preliminary results indicate that LR NGS library preparation method adds valuable information, especially of structural variation, and is likely to improve the diagnostics of neuromuscular disorders.

Factors Affecting Consent Rate for Whole Genome Sequencing of Patients with Congenital Heart Disease

The Whole Genome Sequencing Project initiated by The Hospital for Sick Children of Toronto intents to enroll patients between one to eighteen years of age with congenital heart disease to test the efficiency of such a method for potential personalized diagnoses and treatments. Twenty-five randomly approached patients were chosen to be observed of their consent rate to the project based on factors of age, gender, ethnicity, presence of heart diseases in the patient’s family, and type of congenital heart disease represented. Females and ethnicities not of European descent are considered to be a part of the minority groups, generally being under represented thus having a lower consent rate. There were no significant correlations between consent rates and familial history of heart disease, type of heart disease and age groups. All these findings can aid in future studies of the SickKids Hospital; which patients should be approached for higher consent rates, and the inclusivity of minority groups to alleviate bias in medical studies.

Clinical outcomes after whole-genome sequencing in patients with metastatic non-small-cell lung cancer.

The Personalized Onco-Genomics (POG) program at BC Cancer integrates whole-genome (DNA) and RNA sequencing into practice for metastatic malignancies. We examined the subgroup of patients with metastatic non-small-cell lung cancer (NSCLC) and report the prevalence of actionable targets, treatments, and outcomes. We identified patients who were enrolled in the POG program between 2012 and 2016 who had a tumor biopsy and blood samples with comprehensive DNA (80×, 40× normal) and RNA sequencing followed by in-depth bioinformatics to identify potential cancer drivers and actionable targets. In NSCLC cases, we compared the progression-free survival (PFS) of “POG-informed therapies” with the PFS of the last regimen prior to POG (PFS ratio). In 29 NSCLC cases, 11 were male (38%), the median age was 60.2 yr (range: 39.4–72.6), and histologies included were adenocarcinoma (93%) and squamous cell carcinoma (7%). Potential molecular targets (i.e., cancer drivers including TP53 mutations) were identified in 26 (90%), and 21 (72%) had actionable targets. Therapies based on standard-of-care mutation analysis, such as EGFR mutations, were not considered POG-informed therapies. Thirteen received POG-informed therapies, of which three had no therapy before POG; therefore a comparator PFS could not be obtained. Of 10 patients with POG-informed therapy, median PFS ratio was 0.94 (IQR 0.2–3.4). Three (30%) had a PFS ratio ≥1.3, and three (30%) had a PFS ratio ≥0.8 and <1.3. In this small cohort of NSCLC, 30% demonstrated longer PFS with POG-informed therapies. Larger studies will help clarify the role of whole-genome analysis in clinical practice.

Whole-exome sequencing identifies mutations in FSIP2 as a recurrent cause of multiple morphological abnormalities of the sperm flagella.

Can whole-exome sequencing (WES) of infertile patients identify new genes responsible for multiple morphological abnormalities of the sperm flagella (MMAF)? WES analysis of 78 infertile men with a MMAF phenotype permitted the identification of four homozygous mutations in the fibrous sheath (FS) interacting protein 2 (FSIP2) gene in four unrelated individuals. The use of high-throughput sequencing techniques revealed that mutations in the dynein axonemal heavy chain 1 (DNAH1) gene, and in the cilia and flagella associated protein 43 (CFAP43) and 44 (CFAP44) genes account for approximately one-third of MMAF cases thus indicating that other relevant genes await identification. This was a retrospective genetics study of 78 patients presenting a MMAF phenotype who were recruited in three fertility clinics between 2008 and 2015. Control sperm samples were obtained from normospermic donors. Allelic frequency for control subjects was derived from large public databases. WES was performed for all 78 subjects. All identified variants were confirmed by Sanger sequencing. Relative mRNA expression levels for the selected candidate gene (FSIP2) was assessed by quantitative RT-PCR in a panel of normal human and mouse tissues. To characterize the structural and ultrastructural anomalies present in patients' sperm, immunofluorescence (IF) was performed on sperm samples from two subjects with a mutation and one control and transmission electron microscopy (TEM) analyses was performed on sperm samples from one subject with a mutation and one control. We identified four unrelated patients (4/78, 5.1%) with homozygous loss of function mutations in the FSIP2 gene, which encodes a protein of the sperm FS and is specifically expressed in human and mouse testis. None of these mutations were reported in control sequence databases. TEM analyses showed a complete disorganization of the FS associated with axonemal defects. IF analyses confirmed that the central-pair microtubules and the inner and outer dynein arms of the axoneme were abnormal in all four patients carrying FSIP2 mutations. Importantly, and in contrast to what was observed in patients with MMAF and mutations in other MMAF-related genes (DNAH1, CFAP43 and CFAP44), mutations in FSIP2 led to the absence of A-kinase anchoring protein 4 (AKAP4). The low number of biological samples and the absence of a reliable anti-FSIP2 antibody prevented the formal demonstration that the FSIP2 protein was absent in sperm from subjects with a FSIP2 mutation. Our findings indicate that FSIP2 is one of the main genes involved in MMAF syndrome. In humans, genes previously associated with a MMAF phenotype encoded axonemal-associated proteins (DNAH1, CFAP43 and CFAP44). We show here that FSIP2, a protein of the sperm FS, is also logically associated with MMAF syndrome as we showed that it is necessary for FS assembly and for the overall axonemal and flagellar biogenesis. As was suggested before in mouse and man, our results also suggest that defects in AKAP4, one of the main proteins interacting with FSIP2, would induce a MMAF phenotype. Finally, this work reinforces the demonstration that WES sequencing is a good strategy to reach a genetic diagnosis for patients with severe male infertility phenotypes. This work was supported by the following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 (14-CE15) and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.

Whole-genome sequencing in patients with ciliopathies uncovers a novel recurrent tandem duplication in IFT140.

Ciliopathies represent a wide spectrum of rare diseases with overlapping phenotypes and a high genetic heterogeneity. Among those, IFT140 is implicated in a variety of phenotypes ranging from isolated retinis pigmentosa to more syndromic cases. Using whole-genome sequencing in patients with uncharacterized ciliopathies, we identified a novel recurrent tandem duplication of exon 27-30 (6.7kb) in IFT140, c.3454-488_4182+2588dup p.(Tyr1152_Thr1394dup), missed by whole-exome sequencing. Pathogenicity of the mutation was assessed on the patients' skin fibroblasts. Several hundreds of patients with a ciliopathy phenotype were screened and biallelic mutations were identified in 11 families representing 12 pathogenic variants of which seven are novel. Among those unrelated families especially with a Mainzer-Saldino syndrome, eight carried the same tandem duplication (two at the homozygous state and six at the heterozygous state). In conclusion, we demonstrated the implication of structural variations in IFT140-related diseases expanding its mutation spectrum. We also provide evidences for a unique genomic event mediated by an Alu-Alu recombination occurring on a shared haplotype. We confirm that whole-genome sequencing can be instrumental in the ability to detect structural variants for genomic disorders.

Clinical outcomes after whole genome sequencing in patients with metastatic non-small cell lung cancer.

e20563 Background: The Personalized OncoGenomics (POG) program at the BC Cancer Agency integrates whole genome (DNA) and RNA sequencing into practice for metastatic malignancies. We examine patients with metastatic NSCLC and report the prevalence of actionable targets, treatments, and outcomes. Methods: Between 2012-2016, 217 patients had a tumor biopsy and blood sample with comprehensive DNA (80X; 40X normal) and RNA sequencing followed by in-depth bioinformatics to identify potential cancer “drivers” and/or actionable/treatable targets. In NSCLC cases, we compared the progression-free survival (PFS) of “POG-informed therapies” with the PFS of the last regimen prior to POG (PFS ratio). Results: In 29 NSCLC cases, median age was 60.2 years (range 39.4-72.6), 11 were male (38%), and histologies were: adenocarcinoma (93%); squamous cell carcinoma (7%). Potential molecular targets (i.e. cancer “drivers”) were identified in 26 (90%), and 21 (72%) had actionable targets. 13 received POG-informed therapies, of which 3 had no therapy before POG. Of 10 patients with POG-informed therapy, median PFS ratio was 0.94 (IQR 0.2-3.4). 3 (30%) had a PFS ratio ≥1.3, and 3 (30%) had a PFS ratio ≥0.8 and &lt;1.3. Conclusions: In this NSCLC cohort, 30% demonstrated longer PFS with POG-informed therapies. Larger studies will help clarify the role of whole genome analysis in clinical practice. [Table: see text]

Whole-exome sequencing of familial cases of multiple morphological abnormalities of the sperm flagella (MMAF) reveals new DNAH1 mutations.

Can whole-exome sequencing (WES) of patients with multiple morphological abnormalities of the sperm flagella (MMAF) identify causal mutations in new genes or mutations in the previously identified dynein axonemal heavy chain 1 (DNAH1) gene? WES for six families with men affected by MMAF syndrome allowed the identification of DNAH1 mutations in four affected men distributed in two out of the six families but no new candidate genes were identified. Mutations in DNAH1, an axonemal inner dynein arm heavy chain gene, have been shown to be responsible for male infertility due to a characteristic form of asthenozoospermia called MMAF, defined by the presence in the ejaculate of spermatozoa with a mosaic of flagellar abnormalities including absent, coiled, bent, angulated, irregular and short flagella. This was a retrospective genetics study of patients presenting a MMAF phenotype. Patients were recruited in Iran and Italy between 2008 and 2015. WES was performed for a total of 10 subjects. All identified variants were confirmed by Sanger sequencing. Two additional affected family members were analyzed by direct Sanger sequencing. To establish the prevalence of the DNAH1 mutation identified in an Iranian family, we carried out targeted sequencing on 38 additional MMAF patients of the same geographical origin. RT-PCR and immunochemistry were performed on sperm samples to assess the effect of the identified mutation on RNA and protein. WES in six families identified a causal mutations in two families. Two additional affected family members were confirmed to hold the same homozygous mutation as their sibling. In total, DNAH1 mutations were identified in 5 out of 12 analyzed subjects (41.7%). If we only include index cases, we detected two mutated subjects out of six (33%) tested MMAF individuals. Furthermore we sequenced one DNAH1 exon found to be mutated (c.8626-1G>A) in an Iranian family in an additional 38 MMAF patients from Iran. One of these patients carried the variant confirming that this variant is relatively frequent in the Iranian population. The effect of the c.8626-1G>A variant was confirmed by RT-PCR and immunochemistry as no RNA or protein could be observed in sperm from the affected men. N/A. WES allows the amplification of 80-90% of all coding exons. It is possible that some DNAH1 exons may not have been sequenced and that we may have missed some additional mutations. Also, WES cannot identify deep intronic mutations and it is not efficient for detection of large genomic events (deletions, insertions, inversions). We did not identify any causal mutations in DNAH1 or in other candidate genes in four out of the six tested families. This indicates that the technique and/or the analysis of our data can be improved to increase the diagnosis efficiency. Our findings confirm that DNAH1 is one of the main genes involved in MMAF syndrome. It is a large gene with 78 exons making it challenging and expensive to sequence using the traditional Sanger sequencing methods. We show that WES sequencing is good alternative to Sanger sequencing to reach a genetic diagnosis in patients with severe male infertility phenotypes. This work was supported by following grants: the 'MAS-Flagella' project financed by the French ANR and the DGOS for the program PRTS 2014 and the 'Whole genome sequencing of patients with Flagellar Growth Defects (FGD)' project financed by the Fondation Maladies Rares for the program Séquençage à haut débit 2012. The authors have no conflict of interest.

Clinical application of whole-genome sequencing in patients with primary immunodeficiency

This report illustrates the value of whole genome sequencing (WGS) in elucidating the genetic cause of disease in patients with primary immunodeficiency (PID). As sequencing costs decline, we predict that utilization of next generation sequencing (NGS) in the clinical setting will increase.

Symptom-driven idiopathic disease gene identification.

PurposeRare genetic variants are the major cause of Mendelian disorders, yet only half of described genetic diseases have been causally linked to a gene. In addition, the total number of rare genetic diseases is projected to be far greater than that of those already described. Whole-genome sequencing of patients with subsequent genetic and functional analysis is a powerful way to describe these gene anomalies. However, this approach results in tens to hundreds of candidate disease-causative genes, and the identification of additional individuals suffering from the same disorder can be difficult because of rarity and phenotypic heterogeneity.MethodsWe describe a genetic network–based method to rank candidate genes identified in family-based sequencing studies, termed phenotype informed network (PIN) ranking. Furthermore, we present a case study as an extension of the PIN ranking method in which disease symptoms drive the network ranking and identification of the disease-causative gene.ResultsWe demonstrate, through simulation, that our method is capable of identifying the correct disease-causative gene in a majority of cases. PIN-rank is available at https://genomics.scripps.edu/pin-rank/.ConclusionWe have developed a method to prioritize candidate disease-causative genes based on symptoms that would be useful for both the prioritization of candidates and the identification of additional subjects.

Whole genome sequencing in patients with retinitis pigmentosa reveals pathogenic DNA structural changes and NEK2 as a new disease gene

We performed whole genome sequencing in 16 unrelated patients with autosomal recessive retinitis pigmentosa (ARRP), a disease characterized by progressive retinal degeneration and caused by mutations in over 50 genes, in search of pathogenic DNA variants. Eight patients were from North America, whereas eight were Japanese, a population for which ARRP seems to have different genetic drivers. Using a specific workflow, we assessed both the coding and noncoding regions of the human genome, including the evaluation of highly polymorphic SNPs, structural and copy number variations, as well as 69 control genomes sequenced by the same procedures. We detected homozygous or compound heterozygous mutations in 7 genes associated with ARRP (USH2A, RDH12, CNGB1, EYS, PDE6B, DFNB31, and CERKL) in eight patients, three Japanese and five Americans. Fourteen of the 16 mutant alleles identified were previously unknown. Among these, there was a 2.3-kb deletion in USH2A and an inverted duplication of ~446 kb in EYS, which would have likely escaped conventional screening techniques or exome sequencing. Moreover, in another Japanese patient, we identified a homozygous frameshift (p.L206fs), absent in more than 2,500 chromosomes from ethnically matched controls, in the ciliary gene NEK2, encoding a serine/threonine-protein kinase. Inactivation of this gene in zebrafish induced retinal photoreceptor defects that were rescued by human NEK2 mRNA. In addition to identifying a previously undescribed ARRP gene, our study highlights the importance of rare structural DNA variations in Mendelian diseases and advocates the need for screening approaches that transcend the analysis of the coding sequences of the human genome.