De Novo Mutated TUBB2B Associated Pachygyria Diagnosed by Medical Exome Sequencing and Long-Range PCR

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder caused by mutations in the ABCD1 gene, which encodes an ATP-binding cassette transporter protein, ALDP. The disease is characterized by increased concentrations of very long chain fatty acids (VLCFAs) in plasma, adrenal, testicular, and nerve tissues. For this study, our objective was to conduct clinical, molecular, and genetic studies of a Tunisian patient with X-ALD. The diagnosis was based on clinical indications, biochemical analyses, typical brain-scan patterns, and molecular biology; the molecular analyses were based on PCR, long-range PCR, and sequencing. The molecular analysis by long-range PCR and direct sequencing of the ABCD1 gene showed the presence of a de-novo 2794 bp deletion covering the whole of exon 2. Using bioinformatics tools, we demonstrate that the large deletion is located in a region rich with Alu sequences. Furthermore, we suggest that the AluJb sequence could be the cause of the large deletion of intron 1, exon 2, and intron 2, and the creation of a premature stop codon within exon 3. This report is the first report in which we demonstrate the breakpoints and the size of a large deletion in a Tunisian with X-ALD.

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Whole-exome sequencing with targeted analysis and epilepsy after acute symptomatic neonatal seizures

Pediatric Research ◽

10.1038/s41390-021-01509-3 ◽

2021 ◽

Author(s):

Adam L. Numis ◽

Gilberto da Gente ◽

Elliott H. Sherr ◽

Hannah C. Glass

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

De Novo ◽

List Type ◽

Sequencing Analysis ◽

Neonatal Seizures ◽

Pathogenic Variants ◽

Targeted Analysis ◽

Whole Exome ◽

Epilepsy Gene

Abstract Background The contribution of pathogenic gene variants with development of epilepsy after acute symptomatic neonatal seizures is not known. Methods Case–control study of 20 trios in children with a history of acute symptomatic neonatal seizures: 10 with and 10 without post-neonatal epilepsy. We performed whole-exome sequencing (WES) and identified pathogenic de novo, transmitted, and non-transmitted variants from established and candidate epilepsy association genes and correlated prevalence of these variants with epilepsy outcomes. We performed a sensitivity analysis with genes associated with coronary artery disease (CAD). We analyzed variants throughout the exome to evaluate for differential enrichment of functional properties using exploratory KEGG searches. Results Querying 200 established and candidate epilepsy genes, pathogenic variants were identified in 5 children with post-neonatal epilepsy yet in only 1 child without subsequent epilepsy. There was no difference in the number of trios with non-transmitted pathogenic variants in epilepsy or CAD genes. An exploratory KEGG analysis demonstrated a relative enrichment in cell death pathways in children without subsequent epilepsy. Conclusions In this pilot study, children with epilepsy after acute symptomatic neonatal seizures had a higher prevalence of coding variants with a targeted epilepsy gene sequencing analysis compared to those patients without subsequent epilepsy. Impact We performed whole-exome sequencing (WES) in 20 trios, including 10 children with epilepsy and 10 without epilepsy, both after acute symptomatic neonatal seizures. Children with post-neonatal epilepsy had a higher burden of pathogenic variants in epilepsy-associated genes compared to those without post-neonatal epilepsy. Future studies evaluating this association may lead to a better understanding of the risk of epilepsy after acute symptomatic neonatal seizures and elucidate molecular pathways that are dysregulated after brain injury and implicated in epileptogenesis.

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Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms22041508 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1508

Author(s):

Jordi Maggi ◽

Samuel Koller ◽

Luzy Bähr ◽

Silke Feil ◽

Fatma Kivrak Pfiffner ◽

...

Keyword(s):

Genetic Testing ◽

Long Range ◽

Copy Number Variants ◽

Retinal Disease ◽

Promoter Regions ◽

Long Range Pcr ◽

Cost Efficient ◽

Nucleotide Resolution ◽

Genomic Regions ◽

Next Generation Sequencing Ngs

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

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Analysis workflow to assess de novo genetic variants from human whole-exome sequencing

STAR Protocols ◽

10.1016/j.xpro.2021.100383 ◽

2021 ◽

Vol 2 (1) ◽

pp. 100383

Author(s):

Nicholas S. Diab ◽

Spencer King ◽

Weilai Dong ◽

Garrett Allington ◽

Amar Sheth ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Genetic Variants ◽

De Novo ◽

Whole Exome ◽

Analysis Workflow

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Whole Exome Sequencing in Coloboma/Microphthalmia: Identification of Novel and Recurrent Variants in Seven Genes

Genes ◽

10.3390/genes12010065 ◽

2021 ◽

Vol 12 (1) ◽

pp. 65

Author(s):

Patricia Haug ◽

Samuel Koller ◽

Jordi Maggi ◽

Elena Lang ◽

Silke Feil ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Genetic Screening ◽

De Novo ◽

Efficient Tool ◽

Sequencing Technologies ◽

Whole Exome ◽

Screening And Identification ◽

High Throughput Dna Sequencing ◽

New Mutations

Coloboma and microphthalmia (C/M) are related congenital eye malformations, which can cause significant visual impairment. Molecular diagnosis is challenging as the genes associated to date with C/M account for only a small percentage of cases. Overall, the genetic cause remains unknown in up to 80% of patients. High throughput DNA sequencing technologies, including whole-exome sequencing (WES), are therefore a useful and efficient tool for genetic screening and identification of new mutations and novel genes in C/M. In this study, we analyzed the DNA of 19 patients with C/M from 15 unrelated families using singleton WES and data analysis for 307 genes of interest. We identified seven novel and one recurrent potentially disease-causing variants in CRIM1, CHD7, FAT1, PTCH1, PUF60, BRPF1, and TGFB2 in 47% of our families, three of which occurred de novo. The detection rate in patients with ocular and extraocular manifestations (67%) was higher than in patients with an isolated ocular phenotype (46%). Our study highlights the significant genetic heterogeneity in C/M cohorts and emphasizes the diagnostic power of WES for the screening of patients and families with C/M.

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Optimization and Validation of Multimodular, Long-Range PCR–Based Next-Generation Sequencing Assays for Comprehensive Detection of Mutation in Tuberous Sclerosis Complex

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2020.12.009 ◽

2021 ◽

Author(s):

Sumihito Togi ◽

Hiroki Ura ◽

Yo Niida

Keyword(s):

Next Generation Sequencing ◽

Tuberous Sclerosis ◽

Long Range ◽

Tuberous Sclerosis Complex ◽

Next Generation ◽

Long Range Pcr ◽

Generation Sequencing

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Accurate Molecular Diagnosis of Gaucher Disease Using Clinical Exome Sequencing as a First-Tier Test

International Journal of Molecular Sciences ◽

10.3390/ijms22115538 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5538

Author(s):

Stefania Zampieri ◽

Silvia Cattarossi ◽

Eleonora Pavan ◽

Antonio Barbato ◽

Agata Fiumara ◽

...

Keyword(s):

Exome Sequencing ◽

Molecular Diagnosis ◽

Gaucher Disease ◽

Novel Mutation ◽

Large Deletion ◽

Clinical Exome Sequencing ◽

Number Variation ◽

Long Range Pcr ◽

Set Up ◽

Beta Glucosidase

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.

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Allele-specific long-range PCR/sequencing method for allelic assignment of multiple single nucleotide polymorphisms

Journal of Biochemical and Biophysical Methods ◽

10.1016/s0165-022x(02)00114-8 ◽

2003 ◽

Vol 55 (1) ◽

pp. 1-9 ◽

Cited By ~ 6

Author(s):

Michiyo Nagano ◽

Takahiro Nakamura ◽

Shogo Ozawa ◽

Keiko Maekawa ◽

Yoshiro Saito ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Long Range ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Sequencing Method ◽

Allele Specific ◽

Long Range Pcr

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Whole exome sequencing identifies FOXL2, FOXA2 and FOXA3 as candidate genes for monogenic congenital anomalies of the kidneys and urinary tract

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab253 ◽

2021 ◽

Author(s):

Bixia Zheng ◽

Steve Seltzsam ◽

Chunyan Wang ◽

Luca Schierbaum ◽

Sophia Schneider ◽

...

Keyword(s):

Urinary Tract ◽

Exome Sequencing ◽

Candidate Genes ◽

Whole Exome Sequencing ◽

Congenital Anomalies ◽

De Novo ◽

Horseshoe Kidney ◽

Missense Variant ◽

Whole Exome ◽

Fox Genes

Abstract Background Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the most common cause of chronic kidney disease in the first three decades of life. Variants in four Forkhead box (FOX) transcription factors have been associated with CAKUT. We hypothesized that other FOX genes, if highly expressed in developing kidney, may also represent monogenic causes of CAKUT. Methods We here performed whole exome sequencing (WES) in 541 families with CAKUT and generated 4 lists of CAKUT candidate genes: A) 36 FOX genes showing high expression during renal development, B) 4 FOX genes known to cause CAKUT to validate list A; C) 80 genes that we identified as unique potential novel CAKUT candidate genes when performing WES in 541 CAKUT families, and D) 175 genes identified from WES as multiple potential novel CAKUT candidate genes. Results To prioritize potential novel CAKUT candidates in FOX gene family, we overlapped 36 FOX genes (list A) with list C and D of WES-derived CAKUT candidates. Intersection with list C, identified a de novo FOXL2 in-frame deletion in a patient with eyelid abnormalities and ureteropelvic junction obstruction, and a homozygous FOXA2 missense variant in a patient with horseshoe kidney. Intersection with list D, identified a heterozygous FOXA3 missense variant in a CAKUT family with multiple affected individuals. Conclusion We hereby identified FOXL2, FOXA2 and FOXA3 as novel monogenic candidate genes of CAKUT, supporting the utility of a paralog-based approach to discover mutated genes associated with human disease.

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