Allele-Specific Droplet Digital PCR Combined with a Next-Generation Sequencing-Based Algorithm for Diagnostic Copy Number Analysis in Genes with High Homology: Proof of Concept Using Stereocilin

Early intervention for newborns who are deaf or hard-of-hearing leads to improved language, communication, and social–emotional outcomes. Universal physiologic newborn hearing screening has been widely implemented across the United States with the goal of identifying newborns who are deaf or hard-of-hearing, thereby reducing time to diagnosis and intervention. The current physiologic newborn hearing screen is generally successful in accomplishing its goals but improvements could be made. In the past ten years, genetic testing has emerged as the most important etiological diagnostic test for evaluation of children with deafness and congenital cytomegalovirus has been recognized as a major cause of childhood deafness that may be treatable. A comprehensive newborn hearing screen that includes physiologic, genetic, and cytomegalovirus testing would have multiple benefits, including (1) identifying newborns with deafness missed by the current physiologic screen, (2) providing etiologic information, and (3) possibly decreasing the number of children lost to follow up. We present a framework for integrating limited genetic testing and cytomegalovirus screening into the current physiologic newborn hearing screening. We identify needed areas of research and include an overview of genome sequencing, which we believe will become available over the next decade as a complement to universal physiologic newborn hearing screening.

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“…This type of genetic change cannot be identified using a nucleotide variant detection assay and would require a secondary screening assay. 48…”

Section: Incorporating Genetic Screening Into the Nbhsmentioning

confidence: 99%

A proposal for comprehensive newborn hearing screening to improve identification of deaf and hard-of-hearing children

Shearer

Shen

Amr

et al. 2019

Genetics in Medicine

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“…G-banded chromosome analysis of the chorionic villus sample indicated a normal female karyotype; however, prenatal CMA testing (4x180K CGH + SNP; Agilent Technologies, Santa Clara, CA) detected an apparently homozygous 33.7 kb deletion of chromosome 15q15.3 that included CATSPER2 [reported as arr[GRCh37] 15q15.3(43916972_43950720)x0] (Figure 1). The homozygous loss of CATSPER2 was confirmed by a higher density microarray (244K, Agilent Technologies) and ddPCR (Amr et al, 2018) at intron 7 and exon 7 of CATSPER2; however, parental CMA testing and ddPCR at introns 25 and 23 of STRC indicated that the paternal chromosome 15q15.3 deletion included STRC whereas the maternal chromosome 15q15.3 deletion was inconclusive at the 5' region of STRC due to the absence of copy number probes from exons 1 to intron 22 of STRC and ~6.0 kb upstream of the transcription start site. Due to these inconclusive maternal results, accurate counseling about potential hearing loss in the child could not be provided at that time.…”

Section: To the Editormentioning

confidence: 95%

Prenatal cytogenomic identification and molecular refinement of compound heterozygous STRC deletion breakpoints

Shi

Bai

Kharbutli

et al. 2019

Molec Gen & Gen Med

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Here, we report the prenatal detection of a compound heterozygous deletion at chromosome 15q15.3 by clinical chromosomal microarray (CMA) testing that included the CATSPER2 male infertility gene. However, given the low resolution of CMA at this homologous locus, it was unclear if the neighboring STRC hearing loss gene was also affected. Therefore, we developed a novel allele‐specific PCR strategy, which narrowed the proximal breakpoint of the maternally inherited deletion to a 310 bp interval that was 440 bp upstream from the STRC transcription start site.

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“…Droplet digital PCR (ddPCR) was performed as previously described 12,13 . Given the sequence homology in exons 26 of the C4A and C4B genes, a specific assay (C4BEx26) was designed and optimized to target unique nucleotides in the C4B gene.…”

Section: Droplet Digital Pcr (Ddpcr) and The C4b Assaymentioning

confidence: 99%

The Genomic Landscape of Pediatric Rheumatology Disorders in the Middle East

Fathalla

Alsarhan

Afzal

et al. 2020

Preprint

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Purpose: The goal of this study is to characterize the genomic landscape of pediatric rheumatological disorders in the Middle East, and to assess the clinical utility of genetic findings in this group of patients. Methods: Seventy-one pediatric patients were clinically and genetically assessed for a spectrum of rheumatology-related disease at the only dedicated tertiary childrens hospital in the United Arab Emirates. Clinical genomic investigations included genotyping, NGS-based gene panels, whole exome sequencing, along with rapid sequencing in the intensive care unit (ICU) and urgent setting. Results: The overall positive yield was 46.5% (18.2%-66.7%), while dual diagnoses were made in 2 cases (6%). Although the majority (21/33, 64%) of positive findings involved the MEFV gene, the remaining (12/33, 36%) alterations were attributed to eleven other genes/loci. Copy number variants contributed substantially (5/33, 15.2%) to the overall diagnostic yield. Sequencing-based testing, specifically rapid sequencing, had high positive rate and delivered timely results. Genetic findings guided clinical management plans and interventions in most cases (27/33, 81.8%). We highlight unique findings and provide additional evidence that heterozygous loss of function of the IFIH1 gene increases susceptibility to recurrent fevers. Conclusion: Our study highlights the importance of comprehensive genomic investigations in patients with pediatric rheumatological disorders.

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Allele-Specific Droplet Digital PCR Combined with a Next-Generation Sequencing-Based Algorithm for Diagnostic Copy Number Analysis in Genes with High Homology: Proof of Concept Using Stereocilin

Abstract: NGS-based CNV detection followed by allele-specific ddPCR confirmatory testing is a reliable and affordable approach for copy number analysis in medically relevant genes with homology issues.

Cited by 26 publications

References 25 publications

A proposal for comprehensive newborn hearing screening to improve identification of deaf and hard-of-hearing children

A proposal for comprehensive newborn hearing screening to improve identification of deaf and hard-of-hearing children

Prenatal cytogenomic identification and molecular refinement of compound heterozygous STRC deletion breakpoints

The Genomic Landscape of Pediatric Rheumatology Disorders in the Middle East

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