scholarly journals P0056USE OF CLINICAL EXOME SEQUENCING IN THE DIAGNOSTIC FLOW OF MONOGENIC KIDNEY DISEASES: THE PIEDMONT EXPERIENCE

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Tiziana Vaisitti ◽  
Monica Sorbini ◽  
Martina Callegari ◽  
Silvia Kalantari ◽  
Valeria Bracciamà ◽  
...  

Abstract Background and Aims next-generation sequencing (NGS) technologies are becoming a powerful diagnostic tool in precision medicine. Specifically, exome sequencing can help in the diagnosis of selected diseases, in their medical management and therapeutic choices. Inherited kidney diseases (IKD) are among the major causes for kidney failure, both in children and adults, resulting in increased mortality, high health care costs and need for organ transplantation. In addition, it is worth mentioning that a significant proportion of patients in the kidney transplant lacks a clear diagnosis. This subset of diseases may thus benefit from the application of NGS technology, as the simultaneous investigation of hundreds of genes can lead to the identification of causative variants in a vast population of patients. The aim of this study is to validate the use of a clinical exome sequencing approach in the diagnostic flow for kidney diseases leading to organ failure to i) confirm the clinical diagnosis, ii) find the genetic cause of previously unrecognized diseases and iii) improve the outcome of organ transplantation by excluding live-donors carrying the same mutational burden. Method 160 patients were recruited, directly or following a genetic counseling, exploiting a network of 21 nephrology centers spread across the Piedmont region, coordinated by the “Centro Regionale Trapianti (CRT)” of Torino. Patients were then evaluated for NGS eligibility. DNA extracted from blood samples was checked for integrity, quantified and used for library preparation. A clinical exome sequencing (CES) kit by Illumina was used, allowing for targeted capture, enrichment and sequencing of 6700 clinically relevant genes. Reads were aligned to hg37 reference genome using the Isaac enrichment tool and variants filtered using an ad-hoc set up pipeline of analysis. Results clinical exome sequencing was performed on a diagnostic cohort of 138 patients, both children (37.7%) and adults (62.3%), with a prevalence of male subjects (56.5%). The majority of the cohort (51.5%) presented a positive family history for kidney disease, while 22 patients were excluded from the study as organ failure was most likely the result of secondary events. The cohort was highly heterogeneous with 21% of patients presenting with ciliopathies, 18.1% with glomerular disease, 7.2% with tubular disease while the remaining cohort presented other diseases or was undiagnosed (44.3%). An ad hoc analytical pipeline was designed, based on selected genotype-phenotype correlation database, filter-in metrics, inheritance model and annotation of variants based on public databases and in-silico prediction tools. By adopting well defined criteria of recruitment and analysis, causative genes were identified in 61.6% of cases and in the 57.3% of cases results were in line with the original diagnostic hypothesis. Moreover, 50.8% of cases with organ failure for unknown reasons were solved with the identification of causative genes. Out of the 133 total variants found in the cohort, 63 were classified as pathogenic or likely pathogenic. The remaining 70 identified variants were annotated as variant of unknown significance and will be further investigated. Conclusion Taken together, these results show that CES is a powerful non-invasive tool for the genetic diagnosis of IKD. Identification of disease causative variants may represent a critical step for the diagnosis, clinical management of the patients, and potentially for optimal live-donor selection.

Author(s):  
Tiziana Vaisitti ◽  
Monica Sorbini ◽  
Martina Callegari ◽  
Silvia Kalantari ◽  
Valeria Bracciamà ◽  
...  

Abstract Background A considerable minority of patients on waiting lists for kidney transplantation either have no diagnosis (and fall into the subset of undiagnosed cases) because kidney biopsy was not performed or histological findings were non-specific, or do not fall into any well-defined clinical category. Some of these patients might be affected by a previously unrecognised monogenic disease. Methods Through a multidisciplinary cooperative effort, we built an analytical pipeline to identify patients with chronic kidney disease (CKD) with a clinical suspicion of a monogenic condition or without a well-defined diagnosis. Following the stringent phenotypical and clinical characterization required by the flowchart, candidates meeting these criteria were further investigated by clinical exome sequencing followed by in silico analysis of 225 kidney-disease-related genes. Results By using an ad hoc web-based platform, we enrolled 160 patients from 13 different Nephrology and Genetics Units located across the Piedmont region over 15 months. A preliminary “remote” evaluation based on well-defined inclusion criteria allowed us to define eligibility for NGS analysis. Among the 138 recruited patients, 52 (37.7%) were children and 86 (62.3%) were adults. Up to 48% of them had a positive family history for kidney disease. Overall, applying this workflow led to the identification of genetic variants potentially explaining the phenotype in 78 (56.5%) cases. Conclusions These results underline the importance of clinical exome sequencing as a versatile and highly useful, non-invasive tool for genetic diagnosis of kidney diseases. Identifying patients who can benefit from targeted therapies, and improving the management of organ transplantation are further expected applications.


2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Tiziana Vaisitti ◽  
Monica Sorbini ◽  
Martina Callegari ◽  
Silvia Kalantari ◽  
Valeria Bracciamà ◽  
...  

Abstract Background and Aims Autosomal dominant PKD determines formation of multiple cysts predominantly in the kidneys and usually becomes symptomatic during adulthood and can lead to renal failure. In contrast, in autosomal recessive PKD cysts occur in both the kidneys and the liver and usually presents an earlier onset. Obtaining genetic diagnosis is important to confirm clinical diagnosis and is required before treating with vasopressin 2 receptor blockers, which are the only drugs known to slow down the disease. Furthermore, in the case of kidney transplant from a living family member it is essential to exclude the presence of the mutation in the donor. We used clinical exome sequencing to provide genetic diagnosis to a cohort of patients with a clinical suspicion of PKD. Method 175 patients were referred to the Immunogenetics and Transplant Biology Service of the Turin University Hospital through a network of nephrology centers operating in the Piedmont region. Some patients were referred following genetic counseling. All patients signed an informed consent and the referring physicians provided relevant clinical data. DNA from eligible patients was extracted, checked for integrity, quantified and used for library preparation. A clinical exome sequencing (CES) kit by Illumina was used, allowing the analysis of 6,700 clinically relevant genes. Results Out of the 175 recruited patients eligible for CES, 38 (21.7%) had a clinical suspicion or diagnosis of PKD, with 50% of them presenting family history. The majority of the cohort was represented by male subjects (60.5%) and included both children (34.2%) and adults. The analytical approach was based on initial analysis of genes responsible for PKD (PKD1, PKD2 and PKHD1). If no mutation could be identified, analysis was then extended to a panel of 99 genes responsible for ciliopathies. This approach led to the identification of causative variants in 33/38 (86.8%) of the PKD cohort, while no variant could be identified in 5/38 patients. In 5/33 (15.2%) patients, mutations were inconclusive as found in heterozygosity in genes known to have an autosomal recessive mode of inheritance, while 27/33 (81.8%) were in line with the initial clinical suspicion/diagnosis. Of these, the majority was represented by missense mutations (12), followed by frameshift and nonsense mutations (6 each) and 3 splicing variants. As expected, the majority of mutations were found in PKD1 17/27 (63%), PKD2 3/27 (11.1%) and PKHD1 2/27 (7.4%). In these two latter patients, variants were found as compound heterozygosity. We also found mutations in other genes known to cause cysts, including TSC2 and CPT2. Of note, in 7 patients carrying PKD1 mutations, we found a second variant in PKD1 or PKHD1. Interestingly, when looking at patients characterized by kidney failure but lacking a clinical suspicion at recruitment or diagnosed with other phenotypes (66/175), we found variants in PKD1 and in PKD2 in 11 patients (9 and 2, respectively). Of all identified variants in PKD1, PKD2 and PKHD1 genes, 17.6% were annotated as pathogenic (C5), 41.2% were likely pathogenic (C4) and 41.2% were variants of unknown significance (C3). 19 variants in these genes were not previously reported. All the variants found in genes responsible for PKD were validated and confirmed by Sanger sequencing. Family segregation studies are ongoing. Finally, it is worth mentioning that in a portion of cases (5/38) with clinical and phenotypic features of PKD, supported also by a positive family history, we could not detect mutations in causative genes. These results may be explained by the presence of intronic variants, in line with data reported in literature. Conclusion These results demonstrate that CES may be applied to PKD patients to identify causative variants during their routine diagnostic flow. Furthermore, CES may be a useful tool to detect mutations in PKD-related genes in patients with undiagnosed diseases, considering its rapidly decreasing costs.


2021 ◽  
Vol 22 (11) ◽  
pp. 5538
Author(s):  
Stefania Zampieri ◽  
Silvia Cattarossi ◽  
Eleonora Pavan ◽  
Antonio Barbato ◽  
Agata Fiumara ◽  
...  

Gaucher disease (GD) is an autosomal recessive lysosomal disorder due to beta-glucosidase gene (GBA) mutations. The molecular diagnosis of GD is complicated by the presence of recombinant alleles originating from a highly homologous pseudogene. Clinical exome sequencing (CES) is a rapid genetic approach for identifying disease-causing mutations. However, copy number variation and recombination events are poorly detected, and further investigations are required to avoid mis-genotyping. The aim of this work was to set-up an integrated strategy for GD patients genotyping using CES as a first-line test. Eight patients diagnosed with GD were analyzed by CES. Five patients were fully genotyped, while three were revealed to be homozygous for mutations that were not confirmed in the parents. Therefore, MLPA (multiplex ligation-dependent probe amplification) and specific long-range PCR were performed, and two recombinant alleles, one of them novel, and one large deletion were identified. Furthermore, an MLPA assay performed in one family resulted in the identification of an additional novel mutation (p.M124V) in a relative, in trans with the known p.N409S mutation. In conclusion, even though CES has become extensively used in clinical practice, our study emphasizes the importance of a comprehensive molecular strategy to provide proper GBA genotyping and genetic counseling.


2021 ◽  
Vol 7 (3) ◽  
pp. e597
Author(s):  
Patrick Forny ◽  
Emma Footitt ◽  
James E. Davison ◽  
Amanda Lam ◽  
Cathy E. Woodward ◽  
...  

ObjectiveWe hypothesized that novel investigative pathways are needed to decrease diagnostic odysseys in pediatric mitochondrial disease and sought to determine the utility of clinical exome sequencing in a large cohort with suspected mitochondrial disease and to explore whether any of the traditional indicators of mitochondrial disease predict a confirmed genetic diagnosis.MethodsWe investigated a cohort of 85 pediatric patients using clinical exome sequencing and compared the results with the outcome of traditional diagnostic tests, including biochemical testing of routine parameters (lactate, alanine, and proline), neuroimaging, and muscle biopsy with histology and respiratory chain enzyme activity studies.ResultsWe established a genetic diagnosis in 36.5% of the cohort and report 20 novel disease-causing variants (1 mitochondrial DNA). Counterintuitively, routine biochemical markers were more predictive of mitochondrial disease than more invasive and elaborate muscle studies.ConclusionsWe propose using biochemical markers to support the clinical suspicion of mitochondrial disease and then apply first-line clinical exome sequencing to identify a definite diagnosis. Muscle biopsy studies should only be used in clinically urgent situations or to confirm an inconclusive genetic result.Classification of EvidenceThis is a Class II diagnostic accuracy study showing that the combination of CSF and plasma biochemical tests plus neuroimaging could predict the presence or absence of exome sequencing confirmed mitochondrial disorders.


2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Francisco Martinez-Granero ◽  
Fiona Blanco-Kelly ◽  
Carolina Sanchez-Jimeno ◽  
Almudena Avila-Fernandez ◽  
Ana Arteche ◽  
...  

AbstractMost consensus recommendations for the genetic diagnosis of neurodevelopmental disorders (NDDs) do not include the use of next generation sequencing (NGS) and are still based on chromosomal microarrays, such as comparative genomic hybridization array (aCGH). This study compares the diagnostic yield obtained by aCGH and clinical exome sequencing in NDD globally and its spectrum of disorders. To that end, 1412 patients clinically diagnosed with NDDs and studied with aCGH were classified into phenotype categories: global developmental delay/intellectual disability (GDD/ID); autism spectrum disorder (ASD); and other NDDs. These categories were further subclassified based on the most frequent accompanying signs and symptoms into isolated forms, forms with epilepsy; forms with micro/macrocephaly and syndromic forms. Two hundred and forty-five patients of the 1412 were subjected to clinical exome sequencing. Diagnostic yield of aCGH and clinical exome sequencing, expressed as the number of solved cases, was compared for each phenotype category and subcategory. Clinical exome sequencing was superior than aCGH for all cases except for isolated ASD, with no additional cases solved by NGS. Globally, clinical exome sequencing solved 20% of cases (versus 5.7% by aCGH) and the diagnostic yield was highest for all forms of GDD/ID and lowest for Other NDDs (7.1% versus 1.4% by aCGH) and ASD (6.1% versus 3% by aCGH). In the majority of cases, diagnostic yield was higher in the phenotype subcategories than in the mother category. These results suggest that NGS could be used as a first-tier test in the diagnostic algorithm of all NDDs followed by aCGH when necessary.


2019 ◽  
Vol 35 (2) ◽  
pp. 116-131 ◽  
Author(s):  
Jelena Ruml Stojanovic ◽  
Aleksandra Miletic ◽  
Borut Peterlin ◽  
Ales Maver ◽  
Marija Mijovic ◽  
...  

Clinical exome sequencing is currently being used in diagnostics of various genetic disorders, but studies supporting its application in clinical setting are scarce. The aim of this study was to establish diagnostic and clinical utility of clinical exome sequencing in patients with moderate and severe global developmental delay/intellectual disability. Clinical diagnosis was made in 49 of 88 investigated patients, with overall diagnostic yield of 55.7%. Molecular findings are characterized in detail, including the impact of newly made diagnosis on clinical management. Several previously unreported genotype-phenotype correlations and 33 novel variants are described. Genetic and clinical data were shared through publicly available database. In conclusion, clinical exome sequencing allows identification of causative variants in a significant proportion of patients in investigated clinical subgroup. Compared to whole exome sequencing, it shows similar diagnostic and clinical utility with reduced costs, which could be of particular importance for institutions with limited resources.


Kidney360 ◽  
2021 ◽  
pp. 10.34067/KID.0006902020
Author(s):  
Mina Zamani ◽  
Tahereh Seifi ◽  
Sahar Sedighzadeh ◽  
Samira Negahdari ◽  
Jawaher Zeighami ◽  
...  

This is an Early Access article. Please select the PDF button, above, to view it.


2020 ◽  
Vol 63 (5) ◽  
pp. 103920 ◽  
Author(s):  
Laura Batlle-Masó ◽  
Anna Mensa-Vilaró ◽  
Manuel Solís-Moruno ◽  
Tomàs Marquès-Bonet ◽  
Juan I. Arostegui ◽  
...  

2017 ◽  
Vol 177 (6) ◽  
pp. 485-501 ◽  
Author(s):  
Lucy Shapiro ◽  
Sumana Chatterjee ◽  
Dina G Ramadan ◽  
Kate M Davies ◽  
Martin O Savage ◽  
...  

Background GH insensitivity (GHI) is characterised by short stature, IGF-1 deficiency and normal/elevated serum GH. IGF-1 insensitivity results in pre- and post-natal growth failure with normal/high IGF-1 levels. The prevalence of genetic defects is unknown. Objective To identify the underlying genetic diagnoses in a paediatric cohort with GH or IGF-1 insensitivity using candidate gene (CGS) and whole-exome sequencing (WES) and assess factors associated with the discovery of a genetic defect. Methods We undertook a prospective study of 132 patients with short stature and suspected GH or IGF-1 insensitivity referred to our centre for genetic analysis. 107 (96 GHI, 88 probands; 11 IGF-1 insensitivity, 9 probands) underwent CGS. WES was performed in those with no defined genetic aetiology following CGS. Results A genetic diagnosis was discovered 38/107 (36%) patients (32% probands) by CGS. WES revealed 11 patients with genetic variants in genes known to cause short stature. A further 2 patients had hypomethylation in the H19/IGF2 region or mUPD7 consistent with Silver–Russell Syndrome (total with genetic diagnosis 51/107, 48% or 41/97, 42% probands). WES also identified homozygous putative variants in FANCA and PHKB in 2 patients. Low height SDS and consanguinity were highly predictive for identifying a genetic defect. Conclusions Comprehensive genetic testing confirms the genetic heterogeneity of GH/IGF-1 insensitivity and successfully identified the genetic aetiology in a significant proportion of cases. WES is rapid and may isolate genetic variants that have been missed by traditional clinically driven genetic testing. This emphasises the benefits of specialist diagnostic centres.


2021 ◽  
Vol 12 (1) ◽  
pp. 109-114
Author(s):  
Adrijan Sarajlija ◽  
Slađana Todorović ◽  
Biljana Alimpić ◽  
Maja Čehić

Introduction. Patients affected with Allan-Herndon-Dudley syndrome (AHDS) have a deficiency of monocarboxylate transporter 8 (MCT8), a protein primarily responsible for the transport of triiodothyronine (T3) into the brain. This X-linked disorder affects almost exclusively males with clinical presentation encompassing developmental delay, axial hypotonia, dystonia, poor head control, quadriplegia and absence of speech. Case reports. Patient 1 is a male child referred to a hospital investigation at 11 months due to severe developmental delay and elevated blood ammonia level (163 mcmol/L). Hypotonia and dystonic movements were noted at admission, with facial dysmorphic features. Laboratory findings revealed increased blood lactate (17.2 mmol/L), alanine (533 mcmol/L) and ammonia (391 mcmol/L) concentrations. Serum creatine-kinase levels showed substantial increase over the course of hospitalization up to 6,855 IU/L. Clinical exome sequencing detected a novel hemizygous frameshift insertion c.1456insC in gene SLC16A2, predicted to cause loss of normal protein function either through protein truncation or nonsense-mediated mRNA decay. Segregation genetic testing of the family members revealed that mother, maternal uncle and maternal grandmother carry the same mutation in SLC16A2. The boy`s mother experienced learning difficulties through childhood while maternal uncle is severely affected by AHDS. Patient 2 is a boy referred to clinical geneticist due to severe psychomotor delay of unknown etiology. Moderate serum lactate elevation was the only laboratory abnormality during initial investigations. Diagnosis of AHDS was established by clinical exome sequencing, and subsequent hormonal evaluation revealed increased triiodothyronine (T3) level which corresponds well to genetic diagnosis. Conclusion. Presence of lactic acidosis and/or hyperammonemia in children with severe developmental delay is not specific for inborn disorders of energy production, such as mitochondrial disease. Clinicians should consider thyroid hormones profiling in cases of unexplained severe developmental delay in male children, especially if associated with axial hypotonia and dystonic movements.


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