Diagnostic Genetic Testing in the Neonate

Indications for Testing

When a neonatal phenotype is not fully explained by a nongenetic etiology, indications for genetic testing include:

• Ultrasound anomalies noted in the prenatal period
• Abnormal results of prenatal screening tests
• A newborn with features suggestive of chromosomal aneuploidy or a single gene condition
• Dysmorphic features or congenital anomalies in a newborn
• Dysfunction of unknown etiology in one or more organ systems 
• Neurologic abnormalities, such as seizures  or hypotonia in an infant
• Suggested metabolic abnormality in a newborn
• Suggested specified genetic disorder with a high degree of genetic heterogeneity, which complicates selection of a single definitive test 
• Growth restriction or failure to thrive
• A critical neonatal clinical condition that threatens to be life-limiting
• Family history of a genetic pathogenic variant that may have impact in infancy

Laboratory Testing

Initial Workup

The first step in evaluating a neonate with multiple congenital findings suggestive of a genetic condition is a thorough clinical history, including prenatal history and teratogenic or potential teratogenic exposures, and collection of a three-generation family history, when possible. Parental consanguinity should be assessed. ^,  It is important that the physical evaluation of the newborn includes assessment for dysmorphic features not typical for the patient’s ethnic background or age. ^,  Biochemical studies and/or imaging can be consequential in directing appropriate targeted or comprehensive genetic/genomic testing. Refer to the Methodologies for Neonatal Genetic Evaluation table for descriptions of the relevant genetic tests to consider when suspicion for a genetic condition arises in a neonate.

Diagnosis

For a detailed summary of genetic testing recommendations relevant to a neonatal workup for congenital anomalies, refer to the Diagnostic Genetic Testing in the Neonate algorithm.

Cytogenetic Testing Methods

For neonates suspected of having an underlying chromosomal syndrome (e.g., Down syndrome, Turner syndrome/monosomy X,) it is appropriate to proceed directly to chromosome (karyotype) analysis. Rapid results using FISH are typically an option; FISH screens for the most common chromosomal aneuploidies (often trisomy 13, trisomy 18, trisomy 21, and sex chromosome aneuploidies [in chromosomes X and Y]). FISH aneuploidy screening may yield results within 1-3 days, and standard karyotype analysis on a newborn typically yields a preliminary result in 2-3 days, with a full karyotype report following within 3-7 days, although exact turnaround times may vary. Results of FISH aneuploidy screening studies should be confirmed using standard karyotype analysis when possible. Karyotype analysis may be required to characterize the mechanism for a chromosomal imbalance, such as a Robertsonian translocation causative of trisomy 13 or 21, which is important for reproductive risk counseling.  FISH metaphase analysis may also be used when a high-risk prenatal cell-free DNA screening result or features of a microdeletion/microduplication syndrome are noted (e.g., 22q11.2 microdeletion causative of DiGeorge syndrome). 

CMA is a diagnostic testing option in the newborn period when multiple unexplained congenital anomalies are present, and rapid CMA is available with a decreased turnaround time. CMA is typically performed using a combined copy number variant (CNV)-based and single nucleotide polymorphism (SNP)-based analysis. CMA can detect CNVs (deletions and duplications) and regions of homozygosity (ROH), which may indicate an increased risk for a recessive condition and/or imprinting disorder due to uniparental disomy (UPD). Balanced structural chromosomal imbalances are not detectable by CMA and should be assessed using other cytogenomic methods. ^,  Sequence variants, smaller CNVs, and balanced chromosomal rearrangements including translocations, inversions, insertions, and low-level mosaicism (<20-30%) are not generally detectable by CMA. ^, 

Targeted Genetic Testing

For newborns with a suspected single gene (i.e., Mendelian) disorder, genetic testing using a targeted single gene test or multigene panel may be appropriate (such as for cystic fibrosis, interstitial lung disease, or cardiomyopathies). ^,  There may be other specific testing approaches to consider given the clinical situation, such as for short tandem repeat or imprinting disorders. A downside of targeted genetic testing is that it may be more time intensive, such as when sequential tests are needed to arrive at an answer or multiple tests are unrevealing. Ordering multiple tests can also be more costly in the end when compared with ordering more comprehensive testing.  Targeted testing for an identified familial variant is important to consider when clinical findings are consistent with the gene/variant.

Genomic Sequencing

When an etiology or likely diagnosis is not evident following a thorough preliminary evaluation, genomic sequencing may be appropriate as a first-line test, with or without sequential or concurrent microarray analysis, for a neonate with multiple congenital anomalies. ^, ,  Both WES and WGS are phenotype-driven analyses, and the ability to identify causative variant(s) may be influenced by the quality of the clinical information available for analysis. WES using next generation sequencing (NGS) enables sequencing of the protein-coding regions of the genome (exome), which accounts for 1-2% of the genome itself. ^, ,  The vast majority of pathogenic variants are estimated to be present in the human exome. Exome sequencing is less resource dependent than WGS and may be more cost-effective; thus, it may be selected when a newborn is not at immediate risk of demise.  Exome sequencing will not generally detect intronic variants, methylation defects, trinucleotide repeat disorders, or balanced structural chromosomal rearrangements, such as balanced translocations.  In addition, CNV detection from exome sequencing may vary across laboratories. 

WGS includes sequencing of exon (coding) regions as well as noncoding DNA regions (e.g., introns), which can be important for gene function and regulation.  WGS can be more costly than WES but can offer up to a 10-20% higher diagnostic yield. ^,  Specifically, WGS was found to have a significantly higher yield than WES for neurodevelopmental disorders.  WES or WGS may be appropriately considered when there is not an available targeted gene or panel test or when other testing has been noncontributory. WGS may not detect CNVs in repetitive or difficult to sequence regions of the genome, certain repeat expansions, or methylation abnormalities.  Diagnosis of genetic conditions caused by somatic mosaicism (i.e., certain overgrowth syndromes or lymphatic malformation disorders) by standard WGS or WES may be limited.  Some commercially available WGS tests analyze and report additional information—such as specific CNVs (e.g., for spinal muscular atrophy), repeat expansions, or mitochondrial DNA variants—to further increase the diagnostic yield of the testing. ^,  Genomic sequencing tests may not be available at every institution or clinical setting and may not be approved by insurance payers for any given patient.

For the most accurate results, a trio of samples should be submitted for genomic sequencing: Submit samples from the newborn patient as well as both biological parents, if possible. Trio testing allows for identification of de novo pathogenic variants that can lead to more severe phenotypic outcomes than variants inherited from a parent, and trio testing can provide inheritance information when a compound heterozygous genotype is revealed. ^, ,  Inheritance information is useful for variant phasing and/or variant classification.

Rapid trio analysis using either exome or genome sequencing has value as a first-line test in the neonatal setting when the infant is critically ill. This approach can allow for time-sensitive management and decision-making; results are sometimes available in as few as 3-7 days. Some settings offer ultrarapid genome sequencing, with results in 3 days or fewer.  For rapid genome sequencing, the reporting of VUSs or genes of uncertain significance (GUSs) is generally limited.

Risks of genomic sequencing include the discovery of VUSs or unexpected findings. For individuals undergoing genomic sequencing, the American College of Medical Genetics and Genomics (ACMG) maintains a list of genes for which testing should be offered that can reveal secondary findings—specifically pathogenic/likely pathogenic variants in genes beyond those relevant to the patient’s reported phenotype.  The identification of VUSs or other unexpected findings, such as the identification of nonbiologic relationships or pathogenic variants for adult-onset conditions, is possible.  There may be privacy and insurability issues that arise through comprehensive genetic testing. These possibilities highlight the importance of comprehensive informed consent for families considering genomic sequencing.  Ideally, genetic specialists should be involved in the process of informed consent for these patients/families when possible. 

Genomic sequencing reanalysis can be performed at periodic intervals to reassess patients for causative variants undetectable previously, utilizing improved gene-phenotype associations over time. Given the rapid pace of discovery, reanalysis is important to consider in cases of an elusive diagnosis. ^,  Reanalysis should also be considered when significant new phenotype(s) develop in the patient. Variant reclassification analysis may be available to test informative relatives in an effort to reclassify VUSs, and may be free of charge, depending on the laboratory.