Application Of Whole Exome Sequencing Research Articles

The application of whole-exome sequencing in the early diagnosis of rare genetic diseases in children: a study from Southeastern China.

Genetic diseases exhibit significant clinical and genetic diversity, leading to a complex and challenging diagnostic process. Exploiting novel approaches is imperative for the molecular diagnosis of genetic diseases. In this study, we utilized whole-exome sequencing (WES) to facilitate early diagnosis in patients suspected of genetic disorders. This retrospective analysis included 144 patients diagnosed by singleton-WES Trio-WES between January 2021 and December 2023. We investigated the relevance of diagnosis rates with age, clinical presentation, and sample type. Among the 144 patients, 61 were diagnosed, yielding an overall diagnostic rate of 42.36%, with Trio-WES demonstrating a significantly higher diagnostic rate of 51.43% (36/70) compared to singleton-WES at 33.78% (25/74) (p < 0.05). Global developmental delay had a diagnosis rate of 67.39%, significantly higher than muscular hypotonia at 30.43% (p < 0.01) among different clinical phenotypic groups. Autosomal dominant disorders accounted for 70.49% (43/61) of positive cases, with autosomal abnormalities being fivefold more prevalent than sex chromosome abnormalities. Notably, sex chromosome abnormalities were more prevalent in males (80%, 8/10). Furthermore, 80.56% (29/36) of pathogenic variants were identified as de novo mutations through Trio-WES. These findings highlight the effectiveness of WES in identifying genetic variants, and elucidating the molecular basis of genetic diseases, ultimately enabling early diagnosis in affected children.

Application of Whole-Exome Sequencing in the Prenatal Diagnosis of Foetuses With Central Nervous System Abnormalities.

To investigate the clinical value of whole-exome sequencing (WES) in the diagnosis of foetuses with central nervous system (CNS) abnormalities but having a normal karyotyping and chromosomal microarray result. During the period of 2016-2022, there were a total of 149 foetuses with CNS abnormalities but having negative karyotyping and chromosomal microarray analysis results; WES was performed on these foetuses and their parents. Variants were classified according to ACMG guidelines, and the association of pathogenic variants with specific types of CNS abnormalities was explored. Among these 149 foetuses, three categories of abnormalities, namely, single CNS abnormality, multiple CNS abnormalities, CNS abnormalities along with other organ system abnormalities were identified, for which the detection rate of P/LP variants is 17.4% (12/69), 28.6% (14/49) and 54.8% (17/31), respectively. WES brought about an increase of 28.9% in diagnostic yield in the prenatal evaluation of foetuses with CNS abnormalities but having negative karyotyping and chromosome array results. WES may also be of benefit for the diagnosis of foetuses with isolated CNS abnormalities, as well as for making more informed interpretations of imaging findings and for providing better genetic counselling.

Whole Exome Sequencing of Adult Indians with Apparently Acquired Aplastic Anaemia: Initial Experience at Tertiary Care Hospital

Aplastic anaemia (AA) is a rare hypocellular bone marrow disease with a large number of mutations in the telomerase reverse transcriptase gene (TERT), leading to bone marrow failure. We used our benchmarked whole exome sequencing (WES) pipeline to identify variants in adult Indian subjects with apparently acquired AA. For 36 affected individuals, we sequenced coding regions to a mean coverage of 100× and a sufficient depth was achieved. Downstream validation and filtering to call mutations in patients treated with Cyclosporin A (CsA) identified variants associated with AA. We report four mutations across the genes associated with the AA, TERT and CYP3A5, in addition to other genes, viz., IFNG, PIGA, NBS/NBN, and MPL. We demonstrate the application of WES to discover the variants associated with CsA responders and non-responders in an Indian cohort.

Clinical Diagnosis and Genetic Analysis of Children With Muscular Dystrophies.

More than 90% suspected muscular dystrophy (MD) can be confirmed with multiplex ligation-dependent probe amplification (MLPA) combined with targeted panel, although there are a few that cannot be identified. A total of 312 suspected MD patients were enrolled into the study. The MLPA combined with a targeted myopathy panel were performed. Patients with negative results were subjected to whole exome sequencing (WES), whole genome sequencing (WGS), and/or RNA sequencing (RNA-seq). A total of 275 cases were diagnosed as Duchenne/Becker muscular dystrophy (DMD/BMD) and 20 cases were other types of myopathy or nonmuscular diseases. Six female DMD/BMD patients suffered from varying degrees of typical DMD-like symptoms and 2 others were suspected to be gonadal mosaicism. The systematic application of WES, WGS, and/or RNA-seq highlighted the need for the detection of variants missed by the current standard diagnostic procedures. The identification of female patients and mosaic carriers was crucial to predict the risk of recurrence and allow for optimal genetic counseling.

Application of whole exome sequencing technology in the diagnosis of hereditary eye diseases

Objective: To evaluate the application of whole exome sequencing (WES) in the diagnosis of hereditary eye diseases. Methods: A total of 24 patients who came to the Obstetrics and Gynecology Hospital of Fudan University for reproductive genetic counseling from December 2020 to December 2023 with the main complaint of congenital eye disorders were included in this study. All cases had no known infections or exposure to known teratogenic drugs, karyotype and chromosome microarray analysis (CMA) abnormalities. Genomic DNA was extracted from the peripheral blood of the probands and their family members and tested for WES. Among them, three individual WES and 21 Trio WES were performed. Potential pathogenic sites were screened and analyzed by Sanger sequencing. For RPGRIP1:c.1611+26G>A site, minigene vector was constructed and RT-qPCR was performed to detect the effect of mRNA splicing. Results: A total of 24 families were collected in this study, of which 20 yielded positive results, achieving a diagnosis rate of 83.3% (20/24). The results involved 21 genes and identified 30 distinct variants, 19 of which were new variants reported. Prenatal diagnostic analysis of family 3 revealed that the fetus carried a c.6970G>T heterozygous nonsense mutation in the PRPF8 gene. The results of RT-PCR with the minigene vector at the non-classical splice site in family 24 indicated that the transcription product of the mutant plasmid was partially retained 104 bp in intron 12, resulting in a p.Glu538Valfs*12 alteration of the protein. Conclusions: The high detection rate of WES in the diagnosis of hereditary eye diseases further supports the advantages of its application as an important molecular detection tool for determining the etiology of hereditary eye diseases.

Application of whole exome sequencing in carrier screening for high-risk families without probands.

This study aimed to screen the genetic etiology for the high-risk families including those with an adverse pregnancy history, a history of consanguineous marriages, or a history of genetic diseases, but lack of proband via whole exome sequencing (WES). 128 individuals from high-risk family were tested by WES. The candidate variants were analyzed according to the ACMG criteria to screen the potential carriers. At-risk couples (ARCs) who harbored the same causative gene were provided with precise fertility guidance to avoid the birth of children with birth defects. The total detection rate was 36.72%, with pathogenic/likely pathogenic (P/LP) variants found in 47 individuals, and variants of uncertain significance (VUS) were found in 34. Among couples with adverse pregnancy history: P/LP variants were found in 38 individuals, and VUS were found in 26, for a detection rate of 34.55%; among members of family history of genetic disease or consanguineous marriages: P/LP variants were found in nine individuals, and VUS were found in 8, for a detection rate of 50.00%. Otherwise, we detected 19 ARCs who both carried P/LP variants in the same gene, with a theoretical offspring prevalence of up to 7.42%. In the absence of probands, carrier screening using WES can provide an efficient tool for screening the molecular etiology of high-risk families.

Detection of DNA Contamination in Prenatal Samples from Whole Exome Sequencing Data.

Maternal cell contamination (MCC) in prenatal samples poses a risk for misdiagnosis, and therefore, testing for contamination is necessary during genetic analysis of prenatal specimens. MCC testing is currently performed as a method separate from the diagnostic method. With the increasing application of whole exome sequencing (WES) in prenatal diagnosis, we sought to develop a method to estimate the level of contamination from WES data, aiming to eliminate the need for a separate MCC test. To investigate the impact of MCC on the distribution of the variant allele fraction in WES data, contamination was both simulated in silico and artificially induced. Subsequently, a bioinformatic WES contamination method was developed and validated by comparing its performance to that of the gold standard (short tandem repeat [STR]) MCC test, validated for detecting ≥5% contamination. Finally, post-implementation performance was monitored for a 15-month period. During validation, 270 prenatal samples underwent analysis with both WES and the gold standard test. In 259 samples, the results were concordant (248 not contaminated, 11 contaminated with both tests). In 11 samples, contamination was only detected in WES data (2 of which contained ≥5% contamination with WES, which is above the detection limit of the gold standard test). The data of the post-implementation evaluation on 361 samples, of which 68 were contaminated, were in line with the validation data. Contamination can reliably be detected in WES data, rendering a separate contamination test unnecessary for the majority of samples.

Application of whole exome sequencing in the diagnosis of muscular disorders: a study of Taiwanese pediatric patients.

Muscular dystrophies and congenital myopathies encompass various inherited muscular disorders that present diagnostic challenges due to clinical complexity and genetic heterogeneity. This study aimed to investigate the use of whole exome sequencing (WES) in diagnosing muscular disorders in pediatric patients in Taiwan. Out of 161 pediatric patients suspected to have genetic/inherited myopathies, 115 received a molecular diagnosis through conventional tests, single gene testing, and gene panels. The remaining 46 patients were divided into three groups: Group 1 (multiplex ligation-dependent probe amplification-negative Duchenne muscular dystrophy) with three patients (6.5%), Group 2 (various forms of muscular dystrophies) with 21 patients (45.7%), and Group 3 (congenital myopathies) with 22 patients (47.8%). WES analysis of these groups found pathogenic variants in 100.0% (3/3), 57.1% (12/21), and 68.2% (15/22) of patients in Groups 1 to 3, respectively. WES had a diagnostic yield of 65.2% (30 patients out of 46), detecting 30 pathogenic or potentially pathogenic variants across 28 genes. WES enables the diagnosis of rare diseases with symptoms and characteristics similar to congenital myopathies and muscular dystrophies, such as muscle weakness. Consequently, this approach facilitates targeted therapy implementation and appropriate genetic counseling.

Identification of novel mutations in TPK1 and SLC19A3 genes in families exhibiting thiamine metabolism dysfunction syndrome

Background and aimsThe occurrence of thiamine metabolism dysfunction syndrome (THMD), a rare autosomal recessive condition, may be linked to various mutations found in the TPK1 and SLC19A3 genes. The disease chiefly manifests through ataxia, muscle hypotonia, abrupt or subacute onset encephalopathy, and a decline in developmental milestones achieved during the early stages of infancy. We present findings from an investigation that involved two individuals from Iran, both of whom experienced seizures along with ataxia and hypotonia. The underlying genetic causes were found with the use of next-generation sequencing (NGS) technology, which has facilitated the detection of causal changes in a variety of genetic disorders. Material and methodsThe selection of cases for this study was based on the phenotypic and genetic information that was obtainable from the Center for Comprehensive Genetic Services. The genetic basis for the problems observed among the participants was determined through the application of whole-exome sequencing (WES). Subsequently, sanger sequencing was employed as a means of validating any identified variations suspected to be causative. ResultsThe first patient exhibited a homozygous mutation in the TPK1 gene, NM_022445.4:c.224 T > A:p.I75 N, resulting in the substitution of isoleucine for asparagine at position 75 (p.I75 N). In our investigation, patient 2 exhibited a homozygous variant, NM_025243.4:c.1385dupA:pY462X, within the SLC19A3 gene. ConclusionsCollectively, when presented with patients showcasing ataxia, encephalopathy, and basal ganglia necrosis, it is essential to account for thiamine deficiency in light of the potential advantages of prompt intervention. At times, it may be feasible to rectify this deficiency through the timely administration of thiamine dosages. Accordingly, based on the results of the current investigation, these variations may be useful for the diagnosis and management of patients with THMD.

Preimplantation genetic testing as a means of preventing hereditary congenital myasthenic syndrome caused by RAPSN.

Congenital myasthenic syndrome is a heterogeneous group of inherited neuromuscular transmission disorders. Variants in RAPSN are a common cause of CMS, accounting for approximately 14%-27% of all CMS cases. Whether preimplantation genetic testing for monogenic disease (PGT-M) could be used to prevent the potential birth of CMS-affected children is unclear. Application of WES (whole-exome sequencing) for carrier testing and guidance for the PGT-M in the absence of a genetically characterized index patient as well as assisted reproductive technology were employed to prevent the occurrence of birth defects in subsequent pregnancy. The clinical phenotypes of stillborn fetuses were also assessed. The family carried two likely pathogenic variants in RAPSN(NM_005055.5): c.133G>A (p.V45M) and c.280G>A (p.E94K). And the potential birth of CMS-affected child was successfully prevented, allowing the family to have offspring devoid of disease-associated variants and exhibiting a normal phenotype. This report constitutes the first documented case of achieving a CMS-free offspring through PGT-M in a CMS-affected family. By broadening the known variant spectrum of RAPSN in the Chinese population, our findings underscore the feasibility and effectiveness of PGT-M for preventing CMS, offering valuable insights for similarly affected families.

Application of whole exome sequencing for the inferential analysis of recessive genetic disease carrier status for couples with a child died of Primary immunodeficiency

To explore the value of whole exome sequencing for the inferential analysis of recessive genetic disease carrier status for couples with a child died of Primary immunodeficiency (PID). Clinical data was collected from four couples with a childbearing history of PID who had sought genetic counseling and undergone genetic testing at Henan Provincial People's Hospital from February 2017 to December 2021. Whole exome sequencing (WES) was performed on both partners of each couple, and candidate variants were validated by Sanger sequencing and fluorescent quantitative PCR. Prenatal diagnosis was conducted on fetuses of these couples after confirming the variants. A total of six variants were detected in four genes including IL2RG, BTK, CYBB, and DUOX2. Among these, the c.1265G>A and c.3329G>A variants of the DUOX2 gene and the c.676C>T variant of the IL2RG gene were previously known as pathogenic variants. On the other hand, the Exon5_8del variant of the IL2RG gene, the c.184_185delAC variant of the BTK gene, and the c.472A>T variant of the CYBB gene were unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics, the IL2RG: Exon5_8del, BTK: c.184_185delAC and CYBB: c.472A>T variants were classified as likely pathogenic (PVS1+PM2_Supporting+PP4).Prenatal diagnosis was conducted for three couples during their subsequent pregnancies, and the results revealed that the fetuses had the wild-type genotypes at the c.184_185 position of the BTK gene, the c.472 position of the CYBB gene, and the c.676 position of the IL2RG gene. Follow-up examinations one year after birth has found no abnormality in the infants. WES is an important tool to infer and analyze the carrier status for couples who had given births to children died of PID and improve the positive detection rate.

A novel variation in DEPDC5 causing familial focal epilepsy with variable foci.

Disheveled, EGL-10, and pleckstrin (DEP) domain-containing protein 5 (DEPDC5) is a component of GTPase-activating protein (GAP) activity toward the RAG complex 1 (GATOR1) protein, which is an inhibitor of the amino acid-sensing branch of the mammalian target of rapamycin complex 1 (mTORC1) pathway. GATOR1 complex variations were reported to correlate with familial focal epilepsy with variable foci (FFEVF). With the wide application of whole exome sequencing (WES), more and more variations in DEPDC5 were uncovered in FFEVF families. A family with a proband diagnosed with familial focal epilepsy with variable foci (FFEVF) was involved in this study. Whole exome sequencing (WES) was performed in the proband, and Sanger sequencing was used to confirm the variation carrying status of the family members. Mini-gene splicing assay was performed to validate the effect on the alternative splicing of the variation. A novel variant, c.1217 + 2T>A, in DEPDC5 was identified by WES in the proband. This splicing variant that occurred at the 5' end of intron 17 was confirmed by mini-gene splicing assays, which impacted alternative splicing and led to the inclusion of an intron fragment. The analysis of the transcribed mRNA sequence indicates that the translation of the protein is terminated prematurely, which is very likely to result in the loss of function of the protein and lead to the occurrence of FFEVF. The results suggest that c.1217 + 2T>A variations in DEPDC5 might be the genetic etiology for FFEVF in this pedigree. This finding expands the genotype spectrum of FFEVF and provides new etiological information for FFEVF.

Exome sequencing identifies novel variants associated with non-syndromic hearing loss in the Iranian population.

Autosomal recessive non-syndromic hearing loss (ARNSHL) is a public health concern in the Iranian population, with an incidence of 1 in 166 live births. In the present study, the whole exome sequencing (WES) method was applied to identify the mutation spectrum of NSHL patients negative for GJB2 gene mutations. First, using ARMS PCR followed by Sanger sequencing of the GJB2 gene, 63.15% of mutations in patients with NSHL were identified. Among the identified mutations in GJB2:p.Val43Met and p.Gly21Arg were novel. The remaining patients were subjected to WES, which identified novel mutations including MYO15A:p.Gly39LeufsTer188, ADGRV1:p.Ser5918ValfsTer23, MYO7A: c.5856+2T>c (splicing mutation), FGF3:p.Ser156Cys. The present study emphasized the application of WES as an effective method for molecular diagnosis of NSHL patients negative for GJB2 gene mutations in the Iranian population.

Prenatal whole exome sequencing identified two rare compound heterozygous variants in EVC2 causing Ellis-van Creveld syndrome.

Pathogenic mutations in EVC or EVC2 gene can lead to Ellis-van Creveld (EvC) syndrome, which is a rare autosomal recessive skeletal dysplasia disorder. This study aimed to determine pathogenic gene variations associated with EvC syndrome in fetuses showing ultrasound anomalies. A 32-year-old pregnant woman from Quanzhou, China was investigated. In her pregnancy examination, the fetus exhibited multiple fetal malformations, including a narrow thorax, short limbs, postaxial polydactyly, cardiac malformations, and separation of double renal pelvis. Karyotype, chromosomal microarray analysis and whole exome sequencing were performed for prenatal genetic etiology analysis. Chromosome abnormalities and copy number variants were not observed in the fetus using karyotype and chromosomal microarray analysis. Using whole exome sequencing, two compound heterozygous variants NM_147127.5:c.[2484G>A(p.Trp828Ter)];[871-2_894del] in EVC2 gene were identified in the fetus as pathogenic variants inherited from parents. The study is the first to identify two rare compound variants in EVC2 gene in a Chinese family using whole exome sequencing. The application of whole-exome sequencing would be helpful in fetal etiological diagnosis with ultrasound anomalies.

A novel variant in the QRICH1 gene was identified in a patient with severe developmental delay.

QRICH1 encodes the glutamine-rich protein 1, which contains one caspase activation recruitment domain and is likely to be involved in apoptosis and inflammation. However, the function of the QRICH1 gene was largely unknown. Recently, several studies have reported de novo variants in QRICH1, and the variants have been associated with Ververi-Brady syndrome characterized by developmental delay, nonspecific facial dysmorphism, and hypotonia. Whole exome sequencing, clinical examinations, and functional experiments were performed to identify the etiology of our patient. Here, we added another patient with severe growth retardation, atrial septal defect, and slurred speech. Whole exome sequencing identified a novel truncation variant in the QRICH1 gene (MN_017730.3: c.1788dupC, p.Tyr597Leufs*9). Furthermore, the functional experiments confirmed the effect of genetic variation. Our findings expand the QRICH1 variant spectrum in developmental disorders and provide evidence for the application of whole exome sequencing in Ververi-Brady syndrome.

Whole-Exome Sequencing in Family Trios Reveals De Novo Mutations Associated with Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease characterized by insulin deficiency and loss of pancreatic islet β-cells. The objective of this study is to identify de novo mutations in 13 trios from singleton families that contribute to the genetic basis of T1DM through the application of whole-exome sequencing (WES). Of the 13 families sampled for this project, 12 had de novo variants, with Family 7 having the highest number (nine) of variants linked to T1DM/autoimmune pathways, whilst Family 4 did not have any variants past the filtering steps. There were 10 variants of 7 genes reportedly associated with T1DM (MST1; TDG; TYRO3; IFIHI; GLIS3; VEGFA; TYK2). There were 20 variants of 13 genes that were linked to endocrine, metabolic, or autoimmune diseases. Our findings demonstrate that trio-based WES is a powerful approach for identifying new candidate genes for the pathogenesis of T1D. Genotyping and functional annotation of the discovered de novo variants in a large cohort is recommended to ascertain their association with disease pathogenesis.

A rare compound heterozygous EIF2AK4 mutation in pulmonary veno-occlusive disease

BackgroundPulmonary veno-occlusive disease (PVOD) is a rare, progressive, and oft-fatal condition of pulmonary arterial hypertension that is typically difficult to diagnose and treat. However, with the development of next-generation sequencing technology, an increasing number of patients with PVOD are being diagnosed.MethodsInitially, we used whole exome sequencing (WES) to identify the proband as a rare compound heterozygous mutation of EIF2AK4 in PVOD. Subsequently, the parents of patient underwent EIF2AK4 screening by Sanger sequencing.ResultsIn this study, we describe the family tree of a patient with PVOD with a rare compound heterozygous EIF2AK4 mutation. Moreover, we identified a new EIF2AK4 mutation, c.2236_2237insAAGTCCTTCT, in exon 12 of the proband and his mother. This frameshift mutation led to premature termination of the coding protein sequence and widespread loss of protein function, which promoted the development of PVOD.ConclusionsOur results expand our understanding of the EIF2AK4 mutation spectrum in patients with PVOD, as well as highlight the clinical applicability of WES.

Characterization of a novel androgen receptor gene variant identified in an Iranian family with complete androgen insensitivity syndrome (CAIS): a molecular dynamics simulation study

Androgen insensitivity syndrome (AIS) is a common form of 46, XY disorder in sex development disease (DSD). It is due to the androgen receptor (AR) gene mutations and includes clinical subgroups of complete AIS (CAIS) and partial AIS (PAIS), along with a vast area of clinical heterogeneity of completely normal female external genitalia to male infertility. In this study, the Whole Exome Sequencing (WES) was utilized to detect the cause of DSD in a consanguineous Iranian family with two female patients with normal external genitalia and 46, XY karyotype. Sanger sequencing was applied to validate the candidate variant. Next, we predicted the structural alteration induced by the variant on AR protein using bioinformatics analysis such as molecular dynamic (MD) and molecular docking simulations. WES results identified a novel hemizygous p.L763V variant in the AR gene in the proband that was compatible with the X-linked recessive pattern of inheritance. Bioinformatics studies confirmed the loss of AR function. Based on the American College of Medical Genetics and Genomics (ACMG) guidelines, it was categorized as pathogenic. This study broadens the AR mutation spectrum and introduces the novel p.L763V missense pathogenic variant leading to AR failure to bind to its ligand, and the resulting CAIS clinical subgroup. This study presents a prosperous application of WES and bioinformatics analysis to recognize the underlying cause of DSD in Iran, necessary for its clinical/psychological management. Communicated by Ramaswamy H. Sarma

Predictive genomic tools in disease stratification and targeted prevention: a recent update in personalized therapy advancements.

In the current era of medical revolution, genomic testing has guided the healthcare fraternity to develop predictive, preventive, and personalized medicine. Predictive screening involves sequencing a whole genome to comprehensively deliver patient care via enhanced diagnostic sensitivity and specific therapeutic targeting. The best example is the application of whole-exome sequencing when identifying aberrant fetuses with healthy karyotypes and chromosomal microarray analysis in complicated pregnancies. To fit into today's clinical practice needs, experimental system biology like genomic technologies, and system biology viz., the use of artificial intelligence and machine learning is required to be attuned to the development of preventive and personalized medicine. As diagnostic techniques are advancing, the selection of medical intervention can gradually be influenced by a person's genetic composition or the cellular profiling of the affected tissue. Clinical genetic practitioners can learn a lot about several conditions from their distinct facial traits. Current research indicates that in terms of diagnosing syndromes, facial analysis techniques are on par with those of qualified therapists. Employing deep learning and computer vision techniques, the face image assessment software DeepGestalt measures resemblances to numerous of disorders. Biomarkers are essential for diagnostic, prognostic, and selection systems for developing personalized medicine viz. DNA from chromosome 21 is counted in prenatal blood as part of the Down's syndrome biomarker screening. This review is based on a detailed analysis of the scientific literature via a vigilant approach to highlight the applicability of predictive diagnostics for the development of preventive, targeted, personalized medicine for clinical application in the framework of predictive, preventive, and personalized medicine (PPPM/3 PM). Additionally, targeted prevention has also been elaborated in terms of gene-environment interactions and next-generation DNA sequencing. The application of 3 PM has been highlighted by an in-depth analysis of cancer and cardiovascular diseases. The real-time challenges of genome sequencing and personalized medicine have also been discussed.

Abstract 14506: Re-Evaluating Clinically Relevant Genes in Conotruncal Cardiac Defects by Burden Analyses of Rare and Ultra-Rare Protein-Altering Variants

Conotruncal cardiac defects (CTD) is a group of congenital heart diseases (CHD) involving malformation of outflow tract, with an estimated prevalence of 11.6 per 10,000 births. Currently, a robust consensus on causative genes for nonsyndromic CTD remains to be elucidated. To prioritize clinically relevant genes in whole exome sequencing (WES), we performed gene burden analysis on rare (gnomAD exome allele frequency &lt; 0.01%) protein-altering variants in a cohort of nonsyndromic CTD (n = 245) comparing with ethnicity and location matched controls (n = 853); and expanded to a combined analysis of damaging ultra-rare (CADD ≥ 20 and absent in gnomAD exome) variants involving four CTD cohorts (n = 1451; 391 Chinese and 1060 Europeans) and ethnicity matched gnomAD exome singletons as controls. Gene burden analysis on variants identified within a high-confidence list of 132 CHD genes showed significant (Bonferroni corrected p-value &lt; 0.05) excess of rare protein-altering variants in two well-established genes ( GATA6 , NOTCH1 ) and four potentially relevant genes for CTD ( ANKRD11 , DOCK6 , NPHP4 , STRA6 ). Furthermore, burden analysis of ultra-rare variants in the combined cohorts identified three genes robustly (Bonferroni corrected p-value &lt; 0.05) associated with CTD ( FLT4 , NOTCH1 , TBX1 ). Predominantly more FLT4 loss-of-function variants were reported in Europeans than in Chinese, while GATA6 was the only gene with robust burden in Chinese but was not identified in Europeans, suggesting potential differences in the genetic landscape of CTD disease etiology among different ethnicities. Our results provided evidence of aggregate variant excess for nonsyndromic CTD in case-control comparisons and shortlisted significant genes with potential clinical relevance for further systematic curation, to guide appropriate genetic diagnosis for CTD with the application of WES.