Diagnosis Of Mendelian Diseases Research Articles

Clinical application of next generation sequencing for Mendelian disease diagnosis in the Iranian population

Next-generation sequencing (NGS) has been proven to be one of the most powerful diagnostic tools for rare Mendelian disorders. Several studies on the clinical application of NGS in unselected cohorts of Middle Eastern patients have reported a high diagnostic yield of up to 48%, correlated with a high level of consanguinity in these populations. We evaluated the diagnostic utility of NGS-based testing across different clinical indications in 1436 patients from Iran, representing the first study of its kind in this highly consanguineous population. A total of 1075 exome sequencing and 361 targeted gene panel sequencing were performed over 8 years at a single clinical genetics laboratory, with the majority of cases tested as proband-only (91.6%). The overall diagnostic rate was 46.7%, ranging from 24% in patients with an abnormality of prenatal development to over 67% in patients with an abnormality of the skin. We identified 660 pathogenic or likely pathogenic variants, including 241 novel variants, associated with over 342 known genetic conditions. The highly consanguineous nature of this cohort led to the diagnosis of autosomal recessive disorders in the majority of patients (79.1%) and allowed us to determine the shared carrier status of couples for suspected recessive phenotypes in their deceased child(ren) when direct testing was not possible. We also highlight the observations of recessive inheritance of genes previously associated only with dominant disorders and provide an expanded genotype–phenotype spectrum for multiple less-characterized genes. We present the largest mutational spectrum of known Mendelian disease, including possible founder variants, throughout the Iranian population, which can serve as a unique resource for clinical genomic studies locally and beyond.

Comprehensive genetic analysis reveals the mutational landscape of ABCA4-associated retinal dystrophy in a Chinese cohort

PurposeTo depict the variant profiles of the ABCA4 gene in a large Chinese cohort of patients with ABCA4-associated retinal dystrophy (ABCA4-RD). MethodsWe recruited 290 unrelated Chinese patients with ABCA4-RD and did ABCA4 mutational screening by a combination of Sanger sequencing, targeted exome sequencing, entire ABCA4 locus sequencing, and whole genome sequencing (WGS). The pathogenicity of variants was assessed using in silico tools or in vitro splicing assays following the American College of Medical Genetics and Genomics guidelines. ResultsTwo hundred sixty-eight distinct pathogenic variants were identified, and 57 were novel. In 580 alleles, 22 noncoding region variants outside canonical splice sites and 4 structural variations were found in 44 alleles accounting for 7.6% of all alleles. Bioinformatics analysis showed the complex mechanism of aberrant splicing productsnatural splice site disruption, branch point destruction, and cryptic splice site activation. Correspondingly, minigene assays validated the various abnormal splicing products, including exon skipping, exon elongation, partial exon deletion, and pseudoexon insertion. WGS identified the first inversion variation in ABCA4. ConclusionsThis study systematically depicted the variant profiles of ABCA4 and revealed the missing alleles of patients with ABCA4-RD in a large Chinese cohort. Our findings demonstrated the complexity of molecular diagnosis of Mendelian diseases and the efficiency of WGS for detecting structural variants.

Open-Source Artificial Intelligence System Supports Diagnosis of Mendelian Diseases in Acutely Ill Infants.

Mendelian disorders are prevalent in neonatal and pediatric intensive care units and are a leading cause of morbidity and mortality in these settings. Current diagnostic pipelines that integrate phenotypic and genotypic data are expert-dependent and time-intensive. Artificial intelligence (AI) tools may help address these challenges. Dx29 is an open-source AI tool designed for use by clinicians. It analyzes the patient's phenotype and genotype to generate a ranked differential diagnosis. We used Dx29 to retrospectively analyze 25 acutely ill infants who had been diagnosed with a Mendelian disorder, using a targeted panel of ~5000 genes. For each case, a trio (proband and both parents) file containing gene variant information was analyzed, alongside patient phenotype, which was provided to Dx29 by three approaches: (1) AI extraction from medical records, (2) AI extraction with manual review/editing, and (3) manual entry. We then identified the rank of the correct diagnosis in Dx29's differential diagnosis. With these three approaches, Dx29 ranked the correct diagnosis in the top 10 in 92-96% of cases. These results suggest that non-expert use of Dx29's automated phenotyping and subsequent data analysis may compare favorably to standard workflows utilized by bioinformatics experts to analyze genomic data and diagnose Mendelian diseases.

Clinical exome sequencing findings in 1589 patients

Clinical exome sequencing (CES) is important for the diagnosis of Mendelian diseases, which are clinically and etiologically heterogeneous. Sharing of large amounts of CES data associated with clinical findings will increase the accuracy of variant interpretation. We performed a retrospective study to state the diagnostic yield of CES in 1589 patients with a wide phenotypic spectrum. CES was performed using the Sophia Clinical Exome Sequencing Kit with 4493 genes, followed by sequencing on a NextSeq 500 system. The diagnosis rate was 36.8% when only pathogenic and likely pathogenic variants were included. Consanguineous unions and positive family history were associated with a high diagnostic yield. The neurological disease group had the highest number of patients. The groups with high diagnosis rates were ear, eye, and muscle disease groups. Seven candidate genes (EFHC2, HSPB3, FAAH2, ITGB1, GYG2, CD177, and CSTF2T) that are not yet associated with human diseases were identified. Owing to the high diagnostic yield of CES compared with that of other genetic tests, it can be used as a standard diagnostic test in patients with rare genetic disorders that require a wide differential diagnosis, especially in laboratories with limited resources.

P643: Transcriptome sequencing increases yield of Mendelian disease diagnosis

P643: Transcriptome sequencing increases yield of Mendelian disease diagnosis

Fetal phenotypes of Mendelian disorders: A descriptive study from India.

Exome sequencing (ES)-based diagnosis of Mendelian diseases in the fetus is limited by paucity of phenotypic information. This study reports the comprehensive phenotypes of some fetuses with Mendelian disorders. Next generation technology-based sequencing of all coding regions of the genome (Exome sequencing) or targeted gene sequencing using Sanger or next generation platforms was performed in a cohort of deeply phenotyped, cytogenetically normal fetuses with morphological defects. Prenatal ultrasonographic phenotypes and postmortem details including dysmorphology, histopathology, and radiography were ascertained. Novel candidate genes, novel/unusual findings, and unusual genotypes in cases with confirmed Mendelian disorders are described. Of the 102 fetuses sequenced, 45 (44%) achieved definitive diagnosis of a Mendelian disorder with 50 pathogenic/likely pathogenic variants. The majority (87%) were autosomal recessive, 69% families were consanguineous, and 54% variants were novel. Dysmorphic syndromes, skeletal dysplasias, and metabolic disorders were the commonest disease categories, ciliopathies and dystroglycanopathies, commonest molecular categories. We describe the first fetal description of six monogenic diseases, and nine cases with novel histological findings. Nineteen cases had novel/unusual findings. This cohort demonstrates how deep fetal phenotypes of some Mendelian disorders can show novel/unusual findings, which have important implications for prenatal diagnosis of these conditions.

The importance of automation in genetic diagnosis: Lessons from analyzing an inherited retinal degeneration cohort with the Mendelian Analysis Toolkit (MATK)

PurposeIn Mendelian disease diagnosis, variant analysis is a repetitive, error-prone, and time consuming process. To address this, we have developed the Mendelian Analysis Toolkit (MATK), a configurable, automated variant ranking program. MethodsMATK aggregates variant information from multiple annotation sources and uses expert-designed rules with parameterized weights to produce a ranked list of potentially causal solutions. MATK performance was measured by a comparison between MATK-aided and human-domain expert analyses of 1060 families with inherited retinal degeneration (IRD), analyzed using an IRD-specific gene panel (589 individuals) and exome sequencing (471 families). ResultsWhen comparing MATK-assisted analysis with expert curation in both the IRD-specific gene panel and exome sequencing (1060 subjects), 97.3% of potential solutions found by experts were also identified by the MATK-assisted analysis (541 solutions identified with MATK of 556 solutions found by conventional analysis). Furthermore, MATK-assisted analysis identified 114 additional potential solutions from the 504 cases unsolved by conventional analysis. ConclusionMATK expedites the process of identification of likely solving variants in Mendelian traits, and reduces variability stemming from human error and researcher bias. MATK facilitates data reanalysis to keep up with the constantly improving annotation sources and next-generation sequencing processing pipelines. The software is open source and available at https://gitlab.com/matthew_maher/mendelanalysis.

Improving genetic diagnosis of Mendelian disease with RNA sequencing: a narrative review

Abstract Targeted sequencing and whole exome sequencing are the most common approaches used to detect causative variants in Mendelian diseases; however, using DNA-based sequencing techniques, the current molecular diagnostic yield is at best 50%. In recent years, RNA sequencing has been shown to be able to provide a genetic diagnosis in patients whose conditions were previously unable to be identified by DNA analysis. RNA sequencing can reveal expression outliers, aberrant splicing events, allele-specific expression, and new pathogenic variants, and as such can complement and expand on the traditional genomic methods used to diagnose Mendelian diseases. Therefore, RNA sequencing is expected to become a routine method for genetic diagnosis in the future. This article reviews the applications and challenges of RNA sequencing in the genetic diagnosis of Mendelian diseases.

Identification of novel single-nucleotide variants altering RNA splicing of PKD1 and PKD2.

The development of sequencing techniques identified numerous genetic variants, and accurate evaluation of the clinical significance of these variants facilitates the diagnosis of Mendelian diseases. In the present study, 549 rare single- nucleotide variants of uncertain significance were extracted from the ADPKD and ClinVar databases. MaxEntScan scoresplice is an in silico splicing prediction tool that was used to analyze rare PKD1 and PKD2 variants of unknown significance. An in vitro minigene splicing assay was used to verify 37 splicing-altering candidates that were located within seven residues of the splice donor sequence excluding canonical GT dinucleotides or within 21 residues of the acceptor sequence excluding canonical AG dinucleotides of PKD1 and PKD2. We demonstrated that eight PKD1 variants alter RNA splicing and were predicted to be pathogenic.

Transcriptome Sequencing of Patients With Hypertrophic Cardiomyopathy Reveals Novel Splice-Altering Variants in MYBPC3.

Transcriptome sequencing can improve genetic diagnosis of Mendelian diseases but requires access to tissue expressing disease-relevant transcripts. We explored genetic testing of hypertrophic cardiomyopathy using transcriptome sequencing of patient-specific human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). We also explored whether antisense oligonucleotides (AOs) could inhibit aberrant mRNA splicing in hiPSC-CMs. We derived hiPSC-CMs from patients with hypertrophic cardiomyopathy due to MYBPC3 splice-gain variants, or an unresolved genetic cause. We used transcriptome sequencing of hiPSC-CM RNA to identify pathogenic splicing and used AOs to inhibit this splicing. Transcriptome sequencing of hiPSC-CMs confirmed aberrant splicing in 2 people with previously identified MYBPC3 splice-gain variants (c.1090+453C>T and c.1224-52G>A). In a patient with an unresolved genetic cause of hypertrophic cardiomyopathy following genome sequencing, transcriptome sequencing of hiPSC-CMs revealed diverse cryptic exon splicing due to an MYBPC3 c.1928-569G>T variant, and this was confirmed in cardiac tissue from an affected sibling. Antisense oligonucleotide treatment demonstrated almost complete inhibition of cryptic exon splicing in one patient-specific hiPSC-CM line. Transcriptome sequencing of patient specific hiPSC-CMs solved a previously undiagnosed genetic cause of hypertrophic cardiomyopathy and may be a useful adjunct approach to genetic testing. Antisense oligonucleotide inhibition of cryptic exon splicing is a potential future personalized therapeutic option.

Pitfalls in variant annotation for hereditary cancer diagnostics: The example of Illumina® VariantStudio®

Next Generation Sequencing (NGS), and specifically targeted panel sequencing is the state-of-the-art in clinical genetic diagnosis of Mendelian diseases. However, the bioinformatics analysis and interpretation of the generated data can be challenging. A spotlight on the default transcript selection of a user-friendly, commercially available software that is widely used by genetics professionals, i.e. Illumina® VariantStudio®, is presented. For the sake of comparison, we employed Ensembl VEP, an open-source command-line tool, as it provides flexibility regarding transcript selection. The analysis of NGS data deriving from sequencing of 857 germline DNA samples of cancer patients indicated a concordance of 82.82% between the two software programs. Significantly, using the default transcript configuration of VariantStudio®, we failed to annotate correctly 11.45% of the identified loss-of-function variants. Our results underline the importance of cautious software and transcript selection and the need for reliable, white-box data analysis, along with bioinformatics expertise in clinical diagnostics.

Mapping RNA splicing variations in clinically accessible and nonaccessible tissues to facilitate Mendelian disease diagnosis using RNA-seq

PurposeRNA-seq is a promising approach to improve diagnoses by detecting pathogenic aberrations in RNA splicing that are missed by DNA sequencing. RNA-seq is typically performed on clinically accessible tissues (CATs) from blood and skin. RNA tissue specificity makes it difficult to identify aberrations in relevant but nonaccessible tissues (non-CATs). We determined how RNA-seq from CATs represent splicing in and across genes and non-CATs. MethodsWe quantified RNA splicing in 801 RNA-seq samples from 56 different adult and fetal tissues from Genotype-Tissue Expression Project (GTEx) and ArrayExpress. We identified genes and splicing events in each non-CAT and determined when RNA-seq in each CAT would inadequately represent them. We developed an online resource, MAJIQ-CAT, for exploring our analysis for specific genes and tissues. ResultsIn non-CATs, 40.2% of genes have splicing that is inadequately represented by at least one CAT; 6.3% of genes have splicing inadequately represented by all CATs. A majority (52.1%) of inadequately represented genes are lowly expressed in CATs (transcripts per million (TPM) < 1), but 5.8% are inadequately represented despite being well expressed (TPM > 10). ConclusionMany splicing events in non-CATs are inadequately evaluated using RNA-seq from CATs. MAJIQ-CAT allows users to explore which accessible tissues, if any, best represent splicing in genes and tissues of interest.

Analysis of transcript-deleterious variants in Mendelian disorders: implications for RNA-based diagnostics

BackgroundAt least 50% of patients with suspected Mendelian disorders remain undiagnosed after whole-exome sequencing (WES), and the extent to which non-coding variants that are not captured by WES contribute to this fraction is unclear. Whole transcriptome sequencing is a promising supplement to WES, although empirical data on the contribution of RNA analysis to the diagnosis of Mendelian diseases on a large scale are scarce.ResultsHere, we describe our experience with transcript-deleterious variants (TDVs) based on a cohort of 5647 families with suspected Mendelian diseases. We first interrogate all families for which the respective Mendelian phenotype could be mapped to a single locus to obtain an unbiased estimate of the contribution of TDVs at 18.9%. We examine the entire cohort and find that TDVs account for 15% of all “solved” cases. We compare the results of RT-PCR to in silico prediction. Definitive results from RT-PCR are obtained from blood-derived RNA for the overwhelming majority of variants (84.1%), and only a small minority (2.6%) fail analysis on all available RNA sources (blood-, skin fibroblast-, and urine renal epithelial cells-derived), which has important implications for the clinical application of RNA-seq. We also show that RNA analysis can establish the diagnosis in 13.5% of 155 patients who had received “negative” clinical WES reports. Finally, our data suggest a role for TDVs in modulating penetrance even in otherwise highly penetrant Mendelian disorders.ConclusionsOur results provide much needed empirical data for the impending implementation of diagnostic RNA-seq in conjunction with genome sequencing.

ClinPhen extracts and prioritizes patient phenotypes directly from medical records to expedite genetic disease diagnosis

PurposeDiagnosing monogenic diseases facilitates optimal care, but can involve the manual evaluation of hundreds of genetic variants per case. Computational tools like Phrank expedite this process by ranking all candidate genes by their ability to explain the patient’s phenotypes. To use these tools, busy clinicians must manually encode patient phenotypes from lengthy clinical notes. With 100 million human genomes estimated to be sequenced by 2025, a fast alternative to manual phenotype extraction from clinical notes will become necessary. MethodsWe introduce ClinPhen, a fast, high-accuracy tool that automatically converts clinical notes into a prioritized list of patient phenotypes using Human Phenotype Ontology (HPO) terms. ResultsClinPhen shows superior accuracy and 20× speedup over existing phenotype extractors, and its novel phenotype prioritization scheme improves the performance of gene-ranking tools. ConclusionWhile a dedicated clinician can process 200 patient records in a 40-hour workweek, ClinPhen does the same in 10minutes. Compared with manual phenotype extraction, ClinPhen saves an additional 3–5hours per Mendelian disease diagnosis. Providers can now add ClinPhen’s output to each summary note attached to a filled testing laboratory request form. ClinPhen makes a substantial contribution to improvements in efficiency critically needed to meet the surging demand for clinical diagnostic sequencing.

Clinical sequencing: From raw data to diagnosis with lifetime value.

High-throughput sequencing (HTS) has revolutionized genetics by enabling the detection of sequence variants at hitherto unprecedented large scale. Despite these advances, however, there are still remaining challenges in the complete coverage of targeted regions (genes, exome or genome) as well as in HTS data analysis and interpretation. Moreover, it is easy to get overwhelmed by the plethora of available methods and tools for HTS. Here, we review the step-by-step process from the generation of sequence data to molecular diagnosis of Mendelian diseases. Highlighting advantages and limitations, this review addresses the current state of (1) HTS technologies, considering targeted, whole-exome, and whole-genome sequencing on short- and long-read platforms; (2) read alignment, variant calling and interpretation; as well as (3) regulatory issues related to genetic counseling, reimbursement, and data storage.

Defining categories of actionability for secondary findings in next-generation sequencing

Next-generation sequencing is increasingly used in clinical practice for the diagnosis of Mendelian diseases. Because of the high likelihood of secondary findings associated with this technique, the process of informing...

Genetic Diagnosis of Two Dopa-Responsive Dystonia Families by Exome Sequencing

Dopa-responsive dystonia, a rare disorder typically presenting in early childhood with lower limb dystonia and gait abnormality, responds well to levodopa. However, it is often misdiagnosed with the wide spectrum of phenotypes. By exome sequencing, we make a rapid genetic diagnosis for two atypical dopa-responsive dystonia pedigrees. One pedigree, presented with prominent parkinsonism, was misdiagnosed as Parkinson's disease until a known mutation in GCH1 (GTP cyclohydrolase 1) gene (NM_000161.2: c.631_632delAT, p.Met211ValfsX38) was found. The other pedigree was detected with a new compound heterozygous mutation in TH (tyrosine hydroxylase) gene [(NM_000360.3: c.911C>T, p.Ala304Val) and (NM_000360.3: c.1358G>A, p.Arg453His)], whose proband, a pregnant woman, required a rapid and less-biased genetic diagnosis. In conclusion, we demonstrated that exome sequencing could provide a precise and rapid genetic testing in the diagnosis of Mendelian diseases, especially for diseases with wide phenotypes.

Séquençage des génomes et médecine personnalisée : perspectives et limites.

DNA sequencing technologies have advanced at an exponential rate in recent years: the first human genome was sequenced in 2001 after many years of effort by dozens of international laboratories at a cost of tens of millions of dollars, while in 2013 a genome can be sequenced within 24 hours for a few hundred dollars (exome sequencing takes only a few hours). More and more hospital laboratories are acquiring new high-throughput sequencing devices ("next-generation sequencers", NGS), allowing them to analyze tens or hundreds of genes, or even the entire exome. This is having a major impact on medical concepts and practices, especially with respect to genetics and oncology. This ability to search for mutations simultaneously in a large number of genes is finding applications in the diagnosis of Mendelian diseases (including at birth), routine screening for heterozygotes, and pre-conception diagnosis. NGS is now sufficiently sensitive to analyze circulating fetal DNA in maternal blood (cell-free fetal DNA, cffDNA), enabling applications such as non invasive diagnosis of fetal sex (and X-linked diseases), fetal rhesus among rhesus-negative women, trisomy and, in the near future, Mendelian mutations. Data on multifactorial diseases are still preliminary, but it should soon be possible to identify "strong" factors of genetic predisposition that have so far been beyond the scope of genome-wide association studies (GWAS). In the field of constitutional oncogenetics, NGS can also be used for simultaneous analysis of genes involved in " hereditary " cancers (21 breast cancer genes, 6 colon cancer genes, etc.). More generally, NGS can identify all genomic abnormalities (deletions, translocations, mutations) in a given malignant tissue (hemopathy or solid tumor), and has the potential to distinguish between important mutations (those that drive tumor progression) from " bystander " or accessory mutations, and also to identify "druggable" mutations amenable to targeted therapies (e.g. imatinib and Bcr/Abl rearrangement; verumafemib and the BRAF V600E mutation). Systematic sequencing of all the genes involved in drug metabolism and responsiveness will lead to individualized pharmacogenetics. Finally, sequencing of the tumoral and constitutional genomes, identfication of somatic mutations, and detection of pharmacogenetic variants will open up the era of personalized medicine. The first results of these targeted therapeutic indications show a gain in the duration of remission and survival, although the cost-effectiveness of these approaches remains to be determined. Finally, this huge capacity for genome sequencing raises a number of regulatory and ethical issues.

Diagnóstico prenatal no invasivo: presente y futuro de mano de las nuevas tecnologías

Since the presence of foetal DNA in maternal peripheral blood was demonstrated in 1997, several research groups have developed their activity in this field in order to promote non-invasive prenatal diagnosis into clinical routine.By demonstrating the presence of foetal DNA in the maternal bloodstream, the non-invasive assessment of the sex of the foetus was achieved. This test, together with the foetal RhD determination, has been the only ones incorporated into clinical routine.Although there are research groups working on the diagnosis of Mendelian diseases, efforts have focused on the diagnosis of foetal aneuploidies. At first, the diagnostic scope was limited by the higher presence of maternal DNA co-existing with the foetal DNA. However, recent advances in more sensitive technologies is enabling dramatic progress to be made in this field.

Clinical Application of Genetic Testing

The completion of the human genome project enables us to understand the molecular pathophysiology of human genetic diseases more deeply. In addition, information from genomics has been utilized for the diagnosis of genetic disorders. The technological innovations have been explosive in the field of genetic testing. However, overwhelming genetic information often misleads physicians as well as patients to a wrong belief in the power of genetic testing. Genetic testing implicates many issues such as ethical, legal and social issues. Diagnostic genetic tests can be divided into three primary but overlapping categories: cytogenetic studies (including routine karyotyping, high-resolution karyotyping, and fluorescent in situ hybridization (FISH) studies], biochemical tests, and DNA-based diagnostic tests. DNA-based testing has grown rapidly over the last decade and includes pre- and postnatal genetic testing for the diagnosis of Mendelian diseases in patients, carrier testing, the determination of individual susceptibility to common complex diseases, and population screening of common genetic diseases in a particular population. Theoretically, once the structure, function, and disease association of a gene are well clarified, the clinical application of the genetic testing seems to be feasible. However, the test has to satisfy certain criteria for clinical application at a routine clinical setting; a high sensitivity and positive predictive values, the availability of a controllable quality assurance program, the determination of whether the test is replacing or is complementary to the traditional test, a cost-benefit issue, the possibility of treatment or disease-course modification, possible pre-and postnatal genetic counseling, and so on. In the near future, the application of genetic testing will be further expanded to common diseases and the pharmacogenetic assessment of individuals.