Genetic Variants and Neonatal Disease

Current Genetic Testing Approaches in the Neonatal Intensive Care Unit and their Evolution

Initially, cytogenetic and molecular (DNA-based) genetic testing were considered separate specialties, but with the advent of new sequencing technologies, the boundaries between fields have blurred. Traditionally, cytogenetic tests focused on aneuploidies and large structural chromosomal rearrangements—both balanced and unbalanced—and used techniques such as G-banding karyotype (often referred to clinically as a “karyotype”) and fluorescence in situ hybridization (FISH). The development of chromosomal microarrays (CMA) in the early 2000s maintained the ability to detect aneuploidies and unbalanced chromosomal rearrangements but provided the added ability to detect large regions of homozygosity and smaller copy number abnormalities such as microdeletions and microduplications, and refined the positions of breakpoints for all chromosomal alterations. CMA is still largely applied as a first-tier test in clinical cases of multiple congenital anomalies, developmental delay, intellectual disability, and behavioral differences like autism spectrum disorder. ^, In order to find even smaller insertions and deletions (indels) at the exon level and simultaneously uncover single-nucleotide variants (SNVs), DNA-based sequencing technology is required. Initially, this was accomplished through Sanger sequencing, and later, next-generation sequencing (NGS). In high-resource settings, providers can order these DNA-based tests in the clinical setting as phenotype-specific gene sequencing panels with copy number variant (CNV) analysis, thus merging the realms of cytogenetics and molecular genetics. When the phenotype is both complex and broad, the same technology can be used in a test known as whole exome sequencing (WES) to investigate almost all known genomic exons and a limited number of introns (altogether accounting for approximately 1.5% to 2% of the genome). WES will detect the majority of known disease-causing variant types (SNVs, indels, and CNVs). ^,

WES can be coupled with CMA to ensure fuller coverage of exon-level copy number changes. In the clinical setting, WES can also be ordered simultaneously with sequencing of the patient’s mitochondrial DNA, if indicated. The main purpose of these tests is to investigate for monogenic disorders in which a pathogenic variant or biallelic pathogenic variants confirm a clinical diagnosis. CMA and gene sequence panels currently maintain an active role in the field of neonatology for well-characterized genetic diseases (e.g., autosomal trisomies and microdeletion syndromes), or a phenotypic presentation with a targeted differential diagnosis (e.g., skeletal dysplasias or seizures); in these cases, a precise test may still be an efficient path toward diagnosis, if the expense for WES is prohibitively high and time to result for a center’s currently available WES test platforms remains weeks to months. Another consideration that has become available in addition to the gene sequencing panels based on specific phenotypes and WES is whole genome sequencing (WGS) as a clinical test.

Recognizing the possible prolonged time-to-result and resource-related hurdles of WES and WGS, a study to test the potential for applying a broad sequencing panel to patients with suspected genetic disease was conducted among 20 NICU patients who had been referred to the medical genetics or metabolic inpatient consult services and had features suggesting an underlying genetic or metabolic condition. Twelve infants had been discharged from the NICU, and eight were enrolled prospectively. Subjects underwent a broad genomic sequencing panel identifying sequences of 4813 “disease-relevant” genes that had known associated clinical phenotypes either in OMIM or the United Kingdom’s Human Gene Mutation Database ( www.hgmd.cf.ac.uk/ac/index.php ). The investigators found a diagnostic rate of 40%, suggesting analysis of a broad list of Mendelian genes can produce high diagnostic yields at reduced costs. Of note, only 2 of the 8 infants had genetic diagnoses made by standard clinical approaches inclusive of sequencing of one suspected gene in one patient, and studying a limited panel of 18 sequenced genes in the other. These authors cite another report of 35 infants less than 4 months old in a single tertiary center with suspected genetic conditions in whom 57% had genetic diagnoses from rapid WGS and data analysis. With costs associated with sequencing and data storage decreasing and the speed of performing testing and analyzing results increasing, the field of genomic medicine has been moving toward WES and WGS analyses. The widespread adoption of these technologies has led to gene discovery and novel syndrome characterization and has broadened the phenotypic spectrum of known disorders. The increasing efficiency with which these tests are able to produce a diagnosis has also allowed for individual changes in medical management including earlier administration of targeted therapeutics or enrollment in clinical trials/experimental therapeutics in some instances. While there are many diagnostic and clinical advantages to broader tests such as WES and WGS, aspects to consider and plan for include pretest counseling for secondary and incidental findings, how patient data can be used for data reanalysis and research, or the impact testing could have on future insurance discrimination.