Article Text
Abstract
Recent technological advances have led to the expansion of testing options for newborns with suspected rare genetic conditions, particularly in high-income healthcare settings. This article summarises the key genomic testing approaches, their indications and potential limitations.
- Genetics
- Neonatology
- Ethics
- Technology
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Introduction
Rare genetic conditions are a major cause of neonatal mortality and morbidity.1 2 For some conditions, early recognition allows effective treatment, ranging from dietary modification in phenylketonuria to novel gene therapies in spinal muscular atrophy. For currently untreatable conditions, accurate diagnosis can prevent unnecessary intervention, provide answers for families and allow prenatal counselling for future pregnancies. In high-income healthcare settings, recent technological advances have expanded the available genomic testing approaches for neonates with suspected rare conditions.3 4 Barriers to diagnosis worldwide include inequity in testing infrastructure and a lack of genetic ancestry diversity within genomic research, limiting its clinical applicability to under-represented populations.5 Genomic testing is unique in its potential to reveal information with lifelong consequences for patients and their relatives. Broader testing approaches are not always the most appropriate first-line test, as generating more data increases cost and the risk of inconclusive or unexpected findings. Here, we provide an overview of the key genomic tests for different neonatal indications (see figure 1), alongside the advantages and limitations of each approach.
Clinical assessment
For babies in whom a genetic condition is suspected (see box 1), a thorough clinical assessment is a vital first step.6 A detailed prenatal, birth and postnatal history includes environmental risk factors such as embryotoxins and infection, parental consanguinity and previous pregnancy outcomes. A three-generation family history is represented on a genogram. The physical examination identifies dysmorphic features (features not typical for a child of that age and ethnic background), which should ideally be documented with Human Phenotype Ontology terms, created through international collaboration to enable precise, standardised description of physical features.6 7 Appropriate biochemical and radiological investigations and input from diverse subspecialty teams are key elements of the detailed phenotyping, which underpins genetic diagnosis. If a fetal medicine multidisciplinary team is involved, prenatal genomic testing may have been undertaken and/or postnatal testing may have been planned.
Consider an underlying genetic condition
A baby identified prior to birth due to ultrasound anomalies or abnormalities on prenatal genetic testing.
A baby with features of a monogenic (single gene) condition or chromosomal aneuploidy. A baby with a history of a familial pathogenic variant affecting children.
A baby with congenital anomalies or dysmorphic features (physical features which are not typical for a child of the same age and ethnic background).
A baby with abnormal neurology, features of metabolic disorders or abnormal prenatal/postnatal growth.
A critically unwell baby in whom there is not a clear explanation
Clinical scenario 1
A baby girl is born with swollen hands and feet, low-set ears, widely spaced nipples and a cardiac murmur. As part of her work-up, quantitative fluorescent PCR (QF-PCR) is sent, which confirms Turner syndrome (45,X).
Common aneuploidy testing
Aneuploidy describes additional or missing chromosomes, resulting in a chromosome complement other than 46,XX or 46,XY. Aneuploidy may occur in all cells of the body or only in certain cell lines (mosaicism). The most common aneuploid conditions are Down syndrome (trisomy 21), Edward syndrome (trisomy 18), Patau syndrome (trisomy 13) and sex chromosome aneuploidies such as Turner syndrome (45,X) as in clinical scenario 1. These conditions can be identified through QF-PCR: specific markers on chromosomes 13, 18, 21, X and Y are labelled, amplified and quantified, indicating the number of chromosomal copies in the patient’s sample. QF-PCR is also used in the prenatal setting to diagnose common aneuploidy via amniocentesis or chorionic villus sampling. QF-PCR is relatively cheap and results are usually available within a few days. The limited scope of the test means further testing may be needed.
Karyotyping
Karyotyping is the detailed assessment of chromosome number, size and structure under light microscopy by a cytogeneticist. A karyotype is part of the work-up for babies with ambiguous genitalia and is a follow-up investigation to common aneuploidy testing where low-level mosaicism is suspected or when structural information about the chromosomes is needed. For example, karyotyping identifies uncommon structural causes of aneuploidy such as Robertsonian translocations, which may have a high chance of recurrence in a future pregnancy. Karyotyping is relatively cheap, but for routine indications, it can take several weeks to receive a result.
Microarray
Copy number variations (CNVs) are deletions, duplications or insertions of DNA segments, which may cause a range of congenital anomalies. Microarray analysis screens for CNVs across the genome. Array comparative genomic hybridisation (array CGH) involves adding fluorescently labelled patient and control DNA samples to a slide with thousands of short DNA sequences (probes) from across the genome. The patient and control samples competitively bind to probes: an abnormal ratio of patient to control DNA across consecutive probe regions indicates a CNV at that genomic location. An alternative approach is single-nucleotide polymorphism (SNP) array. An SNP is a nucleotide, where ≥1% of the population has variation; there are millions of SNPs across the genome. Fluorescently labelled patient DNA binds to probes specific to different alleles at known SNPs. Analysis of SNP array data identifies CNVs and can also reveal loss of heterozygosity. This may uncover information about biological relationships, including misattributed parentage, consanguinity or incest.
Alongside common aneuploidy testing, microarray is a first-line test for newborns with multiple congenital anomalies or a suspected CNV, for example, Williams syndrome (7q11.23 microdeletion). Microarray can be performed prenatally if anomalies are detected on ultrasound. A microarray is a very accurate, high-resolution method to identify CNVs as small as 50 kilobases. Microarray analysis is relatively cheap, and when sent urgently, results are usually available within weeks. A microarray does not give structural information about chromosomes, so follow-up karyotyping or other tests such as fluorescence in situ hybridisation may be needed.
Clinical scenario 2
A baby with antenatally diagnosed situs inversus requires intubation and ventilation for severe respiratory distress shortly after birth. After discussion with clinical genetics and respiratory medicine, a respiratory ciliopathies gene panel is sent, which identifies pathogenic loss-of-function variants in both copies of DNAI1, consistent with Kartagener syndrome.
Targeted testing for specific indications
When there is clinical suspicion of a particular condition/group of conditions, a targeted testing strategy may be the best approach. While complex cases benefit from discussion with and assessment by a clinical geneticist, with the mainstreaming of genomic testing, treating teams can increasingly order testing directly. The goal is to improve access to testing and reduce time to diagnosis.8 In the UK, the National Genomic Test Directory details appropriate tests and eligibility criteria for a wide range of indications.9
For conditions with well-established single gene causes such as cystic fibrosis and achondroplasia, the gene in question can be sequenced or known pathogenic variants tested for using a range of approaches. The original sequencing technology is Sanger sequencing, a highly accurate way of determining the nucleotide sequence in a gene, which is then compared with a reference sample to identify pathogenic variants in the patient’s sample. For conditions or phenotypes which may have hundreds or thousands of known associated genes, such as infantile hypotonia or respiratory ciliopathies as in clinical scenario 2, panel testing can be carried out. Historically, all genes in a panel were individually sequenced but now, it is more common for a ‘virtual panel’ to be applied to data generated from whole genome sequencing (WGS) (see the Whole exome and whole genome sequencing section).
Beyond sequencing approaches, other targeted tests include methylation testing for imprinting disorders such as Angelman syndrome and short tandem repeat testing for myotonic dystrophy. In general, if the initial suspected diagnosis is correct, targeted approaches, where available, are an efficient way of reaching a molecular diagnosis. If there is clinical doubt, iterative targeted testing can prolong uncertainty for families, resulting in the so-called ‘diagnostic odyssey’.2
Clinical scenario 3
A preterm baby develops abnormal movements and continuous electroencephalogram telemetry shows multifocal seizures on a background of encephalopathic changes. Seizures are refractory to multiple treatment lines. MRI brain shows reduced cerebral volume. Metabolic investigations and array CGH are normal. Clinical Genetics advise rapid trio WGS which reveals compound heterozygous pathogenic variants in the BRAT1 gene, consistent with a life-limiting encephalopathy and neurodevelopmental disorder. The parents are counselled regarding the poor prognostic outlook and care is re-directed to comfort-focused.
Whole exome and whole genome sequencing
Next-generation sequencing technology allows millions of DNA fragments to be sequenced in parallel, vastly reducing the time and cost of sequencing. Whole exome sequencing (WES) involves sequencing the 1–2% of the genome which is protein-coding (the exome), estimated to contain 85% of disease-causing variants.10 WGS sequences the entire genome, including the non-coding DNA, which has important regulatory functions. For both WES and WGS, variants in the patient sample are identified by comparison with a reference genome and analysed to determine their likelihood of being pathogenic, using the international American College of Medical Genetics and Genomics criteria.11 In the neonatal setting, a trio approach is often used where the baby’s DNA is sequenced alongside their biological parents. This enables the detection of de novo (new) variants in a baby. A de novo variant is more likely to be pathogenic than an autosomal inherited variant, in a baby whose parents do not share the phenotype.
WES or WGS may be performed if initial tests have not yielded a diagnosis or where targeted tests are not available. In the Neonatal Intensive Care Unite (NICU) setting, rapid trio WGS can be performed as a first-line test for critically ill babies in whom a genetic diagnosis could change immediate clinical management, with results available within just 2–3 weeks compared with current time spans of up to a year for non-urgent WGS. The landmark UK 100 000 Genomes Project resulted in a diagnosis for 25% of probands in the pilot analysis of 4660 participants.12 WGS in the NICU setting may lead to higher diagnostic rates of up to 50%.4
The downside of such broad analysis is the frequent identification of variants of uncertain significance without sufficient evidence to be classified as pathogenic or benign, leading to uncertainty for families. These approaches may also identify unexpected findings with health implications, for example, the identification of an adult-onset cancer-susceptibility variant when investigating congenital anomalies in a baby. As with SNP array, trio testing may reveal unknown or concealed information about biological relationships.
As WES generates less data than WGS, it is faster and cheaper.13 Although the non-coding DNA sequenced in WGS may contain pathogenic variants, it is currently difficult to interpret and, therefore, usually excluded from routine analysis.13 Although WES and WGS are highly accurate at identifying sequence changes, other test types may be needed to identify CNVs, structural changes, methylation and repeat expansion disorders.
At present, population newborn screening is limited to a relatively small number of treatable genetic conditions with international variation in screening approaches.14 In England, WGS is being explored as a universal screening approach for newborns to identify treatable, childhood-onset genetic conditions, however potential risks include false positives, leading to unnecessary testing and anxiety for families.15
Family perspectives and bioethical considerations
Pretest counselling facilitates informed consent for genomic testing, which acknowledges potential test outcomes, wider implications and potential limitations (see box 2). In the NICU setting, parents may be sleep-deprived or worried about an unwell infant, which can be barriers to meaningful, informed consent. Families can be supported through a sensitive approach, repeated conversations and measures to support equity in access to testing (see box 3). Genetic Alliance UK’s 2022 ‘good diagnosis’ report identified priorities such as the diagnosis being timely, accurate and collaborative, acknowledging the psychological needs of the whole family.16
Consent for genomic testing
Requires written consent from a person with parental responsibility.
The consent discussion should cover:
The indications, benefits and limitations of testing.
The possible outcomes of testing including finding a diagnosis, not finding a diagnosis or identifying findings of uncertain significance.
The possibility of incidental/secondary findings with implications for health or biological relationships.
Issues surrounding data storage and data use for audit and research.
That consent can be withdrawn at any time.
Plan for return of results and support from clinical genetics and/or genetic counsellors.
Equality, diversity and inclusion (EDI) in newborn genomic testing
Alongside systemic changes to improve diversity within genomic research and policy, at the patient-facing level, EDI can be promoted19 .
Ethnicity: measures include availability of interpreters, written information in a range of languages and collaboration with cultural, community or religious leaders.
Disability: dominantly inherited genetic conditions can cause disability in parents—measures include sign language interpreters, large print/Braille/audio information and individualised support for neurodiverse parents or those with learning disabilities.
LGBTQIA+ inclusivity: consider different family structures including conception with donor gametes, which may preclude trio testing. When representing transgender people on genograms, their gender identity and sex assigned at birth should both be annotated20 .
Studies of family experiences with genomic testing have found positive overall perceptions of utility. In a US study of parents whose babies had received rapid genomic testing in NICU, 97% of parents felt it was ‘useful’ or ‘somewhat useful’ despite only 23% of infants receiving a diagnosis.17 Families reported that even a ‘negative’ result helped them feel more knowledgeable about their child’s health and better informed for future reproductive planning. It should be noted that for conditions which are life limiting or result in significant disability, receiving a diagnosis within the first days of the baby’s life may be very traumatic for the parents and disrupt early parent–child bonding.18 Alongside referral to Clinical Genetics and appropriate specialist teams, there are many sources of information and support for families following a genetic diagnosis.
Conclusion
Genomic testing of neonates is key for the early diagnosis of rare, genetic conditions. Beyond diagnosis of symptomatic infants, it is possible that population-based newborn screening with WGS will become a reality in the not too distant future. To optimise care for babies with genetic conditions, the neonatal workforce must be equipped with a comprehensive understanding of the available technology as well as the potential ethical and emotional implications for families.
Clinical bottom line
Rare genetic conditions are a major cause of neonatal mortality and morbidity.
The best testing strategy depends on the clinical presentation. Less expensive, faster tests such as quantitative fluorescent PCR and microarray are often the best first-line tests.
Whole exome sequencing and whole genome sequencing are now options in high-resource healthcare settings and may lead to a diagnosis where other testing strategies have not but have risks of inconclusive and unexpected findings.
There is global inequity in access to genomic testing and there are many ways to promote equality, diversity and inclusion in daily clinical practice.
Ethics statements
Patient consent for publication
Ethics approval
Not applicable.
References
Footnotes
Correction notice This paper has been updated since it was first published. Dr Ally Mintoft's surname has been corrected.
Contributors The article was conceived by JS and AM. JS and AM wrote the manuscript with supervision and critical feedback from GK and TA.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer-reviewed.