Whole Exome Sequencing In Diagnosis Research Articles

The Role of Whole-Exome Sequencing in Diagnosis of Genetic Disorders: A Case Report of a Novel Mutation in the Fibrinogen A-Alpha Chain Gene

The Role of Whole-Exome Sequencing in Diagnosis of Genetic Disorders: A Case Report of a Novel Mutation in the Fibrinogen A-Alpha Chain Gene

Genetic variations in the DYNC2H1 gene causing SRTD3 (short-rib thoracic dysplasia 3 with or without polydactyly).

Background and aims: Short-rib thoracic dysplasia 3 with or without polydactyly (SRTD3) represents a type of severe fetal skeletal dysplasia (SD) characterized by shortened limbs, narrow thorax with or without polydactyly, which is caused by the homozygous or compound heterozygous mutations in the DYNC2H1 gene. SRTD3 is a recessive disorder, identification of the responsible genetic variation would be beneficial to an accurate prenatal diagnosis and well-grounded counseling for the affected families. Material and methods: Two families having experienced recurrent fetal SDs were recruited and submitted to a multiplatform genetic investigation. Whole-exome sequencing (WES) was performed with samples collected from the probands. Sanger sequencing and fluorescent quantitative PCR (qPCR) were conducted as validation assays for suspected variations. Results: WES identified two compound heterozygous variations in the DYNC2H1(NM_001080463.2) gene, namely c.2386C>T (p.Arg796Trp) and c.7289T>C (p.Ile2430Thr) for one; and exon (64-83)del and c.8190G>T (p.Leu2730Phe) for the other, respectively. One variant in them, exon (64-83)del, was novelly identified. Conclusion: The study detected two compound heterozygous variation in DYNC2H1 including one novel deletion: exon (64-83) del. Our findings clarified the cause of fetal skeletal dysplasia in the subject families, provided guidance for their future pregnancies, and highlighted the value of WES in diagnosis of skeletal dysplasia with unclear prenatal indications.

Whole exome sequencing for diagnosis of hereditary thrombocytopenia.

Hereditary thrombocytopenia comprises extremely diverse diseases that are difficult to diagnose by phenotypes alone. Definite diagnoses are helpful for patient (Pt) management.To evaluate the role of whole exome sequencing (WES) in these Pts.Cases with unexplained long-standing thrombocytopenia and/or suggestive features were enrolled to the observational study. Bleeding scores and blood smear were evaluated. The variant pathogenicity from WES was determined by bioinformatics combined with all other information including platelet aggregometry, flow cytometry, and electron microscopy (EM).Seven unrelated Pts were recruited. All were female with macrothrombocytopenia. Clinical bleeding was presented in four Pts; extra-hematological features were minimal and family history was negative in every Pt. WES successfully identified all the 11 responsible mutant alleles; of these, four have never been previously reported. Pt 1 with GNE-related thrombocytopenia showed reduced lectin binding by flow cytometry, increased glycogen granules by EM and a novel homozygous mutation in GNE. Pts 2 and 3 had phenotypic diagnoses of Bernard Soulier syndrome and novel homozygous mutations in GP1BB and GP1BA, respectively. Pt 4 had impaired microtubule structures, concomitant delta storage pool disease by EM and a novel heterozygous TUBB1 mutation. Pt 5 had sitosterolemia showing platelets with reduced ristocetin responses and a dilated membrane system on EM with compound heterozygous ABCG5 mutations. Pts 6 and 7 had MYH9 disorders with heterozygous mutations in MYH9.This study substantiates the benefits of WES in identifying underlying mutations of macrothrombocytopenia, expands mutational spectra of four genes, and provides detailed clinical features for further phenotype-genotype correlations.

Whole-exome sequencing for diagnosis of Peters-plus syndrome after prenatal diagnosis of recurrent low PAPP-A and multiple fetal anomalies in two consecutive pregnancies.

We report a case of two consecutive pregnancies in the same couple presenting with very low pregnancy-associated plasma protein A (PAPP-A), with both pregnancies affected by multiple anomalies of a similar phenotype identified during mid-trimester ultrasound, and eventual diagnosis of Peters-plus syndrome. This case is important in expanding the differential for very low PAPP-A. It also demonstrates the diagnostic value of whole-exome sequencing (WES) after prenatal diagnosis of recurrent fetal ultrasonographic findings. The importance and complexity of providing patient education to enable informed consent for next generation sequencing technologies is discussed.

Application of Whole Exome Sequencing in Diagnosis of Pediatric Acute Liver Failure of Unknown Etiology

Application of Whole Exome Sequencing in Diagnosis of Pediatric Acute Liver Failure of Unknown Etiology

Effect of whole exome sequencing in diagnosis of inborn errors of metabolism and neurogenetic disorders: A review articles

Background: Inborn errors of metabolism are complex disorders with huge variety in clinical manifestations. Metabolic disorders have overlapping clinical pictures with other genetic disorders especially neurogenetic diseases. It causes hardship in diagnosis of metabolic and neurogenetic diseases in some occasions. There is growing rise for using whole exome sequencing in diagnosis of genetic disease during recent years result from decreasing its cost. Methods: We reviewed articles by search in medline database to evaluate impact of whole exome sequencing in diagnosis of metabolic and neurogenetic diseases in clinic field. Results: Review declared whole exome sequencing is effective in recognizing metabolic and neurologic diseases especially in complex cases. Diagnostic yield of whole exome sequencing in identifying metabolic and neurogenetic disease has large variety, ranging from 16% to 68% that increase during recent years. It has ability to change patients’ management in 2-44% of cases depend on different studies. Whole exome sequencing can provide new information about new disease, new variants and phenotypes. Careful interpretation of data obtained by WES and evaluation of correlation between clinical manifestation and results is necessary for clinical application of this technology. Conclusions: Whole exome sequencing is useful way for diagnosis of metabolic and neurogenetic diseases essentially in puzzling cases.

Importance of whole exome sequencing in diagnosis of diseases (the Libyan experience)

The exome is the part of the genome formed by exons (Figure 1); the sequences which when transcribed remain within the mature RNA after introns are removed by RNA splicing. The exon covers between 1 and 2% of the genome, depending on species [1]. Whole-exome sequencing (WES) is a revolutionary technique, which targets the protein-coding regions of the genome and has proven success in identifying new causal mutations and major congenital anomalies for diseases of previously unknown aetiology. With a successful diagnostic rate approaching 25% for rare disease, its clinical utility is becoming increasingly popular [3]. WES consists in the capture, sequencing and analysis of all exons of all protein-coding genes in the human genome. Instead of analysing the whole genome, composed of roughly three billion base-pairs, WES focuses only on approximately 30 million base-pairs which are translated into functional proteins. It is believed that the mutations of genes responsible for the formation of the functional proteins are the most likely to have a severe direct phenotypic consequence [4]. Therefore, WES was suggested to be much less costly and more efficient method of identifying all possible mutations in genes, compared to other methods such as genome-wide association studies or whole-genome sequencing (WGS) [4]. Before the discovery of WES, many patients with genetic diseases were not given a specific diagnosis. The lack of a diagnosis can have considerable adverse effects for patients and their families, including failure to identify potential treatments, failure to recognize the risk of recurrence in subsequent pregnancies, and failure to provide anticipatory guidance and prognosis [5]. Therefore, WES is extensively used nowadays to diagnose novel diseases and find novel causative mutations for known disease phenotypes. We in our paediatric neurology unit in Tripoli Children Hospital, serving the west and south area of the country with a population of 1.5 million children below 15 years of age, are faced almost on a daily basis with patients of developmental delay difficult to diagnose in spite of doing the basic neuro-metabolic screen and imaging. Since 2010, our unite is cooperating with the research center of Dr. Joseph Gleeson laboratory for paediatric brain diseases, Howard Hughes Medical Institute, University of California San Diego USA. We were able to send blood from 100 families whom they have at least two affected children without a clear diagnosis, and we were able to find out the causative gene in about 60% and we discovered about 10 novel genes, four of them have been published in highly ranked international journals [6,7,8]. When a researcher is faced with a novel gene variant, he/she needs to prove that this gene mutation is pathological. This prove requires a lengthy testing procedures by doing functional testing on knocked-down mouse or zebra fish for the specific mutant gene. Unfortunately, this kind of research is lacking in Libya and we should put all needed efforts to establish these techniques in our research laboratories. Moreover, the researchers in Dr Gleeson Laboratory now started to study WES negative patients by doing WGS, which mean to study the rest of 99% of the genome that don’t code for protein and performing sequencing of RNA from cell line skin fibroblast from these families. By comparing the sequencing and levels of RNA from patient’s cells we can solve more cases of patients with genetic defects.

Utility of Whole Exome Sequencing in Diagnosis of Pediatric Platelet Disorders: A Subanalysis of the Pediseq Study

Background: Inherited Platelet Disorders (IPD) are individually rare disorders that have many different molecular causes. Diagnosis of IPD is often complicated by the need for complex testing that is not readily available at many centers and the lack of available testing to define the molecular cause of some disorders. While some platelet disorders are sufficiently defined by functional characterization, recent data suggests that some platelet disorders may predispose to significant other complications including cancer predisposition, myelofibrosis or hearing loss. Therefore, it may be important to establish a molecular diagnosis to better counsel families about necessary follow up and possible risks. The goal of this study was to determine the diagnostic yield of whole exome sequencing in a cohort of 22 pediatric patients with clinical presentation suggesting an underlying genetic cause (or positive family history of platelet disorder with no prior genetic diagnosis).Methods: Peripheral blood was collected from patients identified as likely to have an inherited platelet disorder after informed consent. Samples were also obtained from parents and siblings for co-segregation and variant calling. Genomic DNA was extracted manually using the Gentra Puregene Blood Kit. Exome capture was performed using the Agilent SureSelect v4 and 100 base paired end sequencing was done on an IlluminaHiSeq 2000 with 100X average coverage. Sequencing reads were generated in FASTQ format and mapped to human genome GRCh37 (hg19) and Novoalign v2.08 was used for optimal alignment. Disease-related variants were extracted from HGMD to identify variants that might be missed. Variant filtering and pathogenicity classification was performed using a customized pipeline and manual curation. We identified 53 genes of interest and on average across all exomes with an indication for a platelet disorder, bases were sequenced at a minimum depth of 15X to be considered covered within an exon. 80.4% of exons were 100% covered with this technology completely, while 10.4% of exons were partially covered (>40 to <100% bases) and 9.2% of exons were not covered (<40% of bases covered at a minimum of 15X depth).Results: 22 patients were enrolled over a 12-month period. Overall, 82% of patients had variants identified in platelet related genes on whole exome sequencing with 64% of patients returning at least one variant of uncertain significance (14) and 23% (5) patients returning definite positive results. One patient referred for further work up carried the initial diagnosis of ITP, but had macrothrombocytopenia since early childhood and bleeding out of proportion to the platelet count. Flow cytometry and functional studies performed on referral suggested possible Bernard Soulier Syndrome and sequencing confirmed homozygous pathogenic mutation in GP9. One patient with congenital thrombocytopenia and history of intracranial hemorrhage with a similarly affected sibling had confirmed pathogenic MYH9 mutation, allowing clinicians to offer prenatal diagnosis during a third pregnancy. One patient with a significant bleeding phenotype was a compound heterozygote for two novel RASGRP2 variants, but the functional significance of those variants is uncertain and further studies are underway to determine whether these variants are causative. 18% of patients (4) had negative sequencing results (no reportable variants in platelet related genes identified). Conclusions: Whole exome sequencing can be a powerful diagnostic tool in identifying the molecular cause of disease in a cohort of patients with suspected inherited platelet disorders. The majority of patients, however, will receive results of uncertain significance and centers that undertake this testing will require an infrastructure to allow for further functional evaluation, which will help in reclassification of these variants, and ensure that results are correctly interpreted. Clinicians who undertake ES to diagnose IPD need to understand limitations of the test as well as the full significance of results that may be returned. DisclosuresLambert:Novartis: Consultancy.