Newborn screening (NBS) program refers to a nation-wide or state-wide program that identifies and treats newborns with
rare congenital conditions before the onset of symptoms, preventing premature death and serious disability
in thousands of newborns. Following the great success of Next Generation Sequencing (NGS) technology
in the clinical diagnosis of genetic disorders, a lot of expectations have been raised among researchers,
clinicians and the public for its implementation in the newborn screening program (NBS). But in view of the
ethical, legal and social issues revolving around the use of genome sequencing approaches in health-care and
public health programs it is necessary to address these issues beforehand to avoid its long term failure. This
review will focus on the realized and expected benefits of using NGS in state NBS program and will also
highlight the major hurdles and practical difficulties that have to be considered for materialization of such a
program.
Till date Sanger sequencing has been the gold standard for DNA sequencing. Using this technology a major foray called
the Human Genome project started in 1990 and lasted for 13 long years wherein $3 billion was expended to determine the
whole human genome sequence. But in spite of the known usefulness of DNA sequence analysis at that time it was
beyond imagination for the clinicians to think about sequencing every patient’s genome to find possible
variants underlying the concerned disease due to practical limitations of this technology i.e. being expensive
and time consuming. Thereafter, with continuous advancement in the research methodology and scientific
aptitude, DNA sequencing underwent major improvements making it possible to sequence a large number of
samples in parallel which was not quite possible by Sanger sequencing. The emergence of Next generation
sequencing (NGS) in 2005 met the key shortcomings of Sanger sequencing in being more cost effective,
rapid, and requiring lesser amount of DNA. Clinical implementation of NGS for disease characterization in
individual patients was found to be highly fruitful (Worthey et. al., 2011; Lupski et. al., 2010; Liew et. al.,
2013).
NGS, in view of its present achievements in the field of diagnosis, has heightened the expectations of the scientific
community and clinicians in incorporating it in routine clinical practice and more recently in mass screening programs like
NBS (new born screening).This review will focus on the realized and expected benefits of using NGS in the state NBS
program and will also highlight the major limitations that have to be considered for materialization of such a
program.
NBS is an essential, preventive public health program established internationally in order to identify disorders in
newborns that was started almost 55 years ago. It began as a method for pre-symptomatic diagnosis and preventive
treatment for one disorder Phenylketonuria (PKU) in newborns and later, in the 1990s, with the introduction of a much
cheaper and reliable analytical technique Tandem mass spectrometry, more than 30 different metabolic disorders were
added to the screening panel in neonates which led to a significant expansion of NBS. The initial guidelines followed for
including any disorder in the neonatal screening program were based on the Wilson and Jungner criteria (Laine et.
al., 2013) that emphasized on conditions that are considered as an important health problem with well
understood natural history and requiring immediate medical intervention in order to prevent serious and
permanent illness, and for which there is an available treatment. Currently with expanded NBS most babies are
screened at birth for between 30 and 50 genetic disorders, primarily by using tandem mass spectrometry
(MS/MS) and many of these disorders do not fit into the classical paradigm of NBS. In this way, newborns
are being screened even for conditions that do not present as emergencies and may not be immediately
life-threatening, but could benefit from treatment with prophylactic antibiotics or if their screening might
have additional benefit to parents for reproductive purposes (Grosse et. al., 2006). Thus, right from the
beginning, expansion of NBS has been an attempt to provide maximum benefit to the child and the family,
through use of the growing knowledge about genetic disorders and technology. Now when we are in the
Genomic era where progress is taking place in an ever quickening pace, many of the seemingly unrealistic
visions are beginning to materialize. The recent reduction in the cost and time required for sequencing
the whole genome and the promise it holds both for research and health care have drawn a significant
momentum around the idea of using NGS in a state-run mass screening program for NBS. But before this
thought could be objectified we must recognize all the challenges that might interfere in attaining this
vision.
NBS, as discussed before, is a state-run mass screening program that aims to identify serious, treatable disorders in
asymptomatic newborns that require immediate medical intervention. To carry out newborn screening NGS can be used
in a better way. Being a high throughput technology it can scan the entire sequence of the newborn’s genome to produce a
huge amount of information about target and off target disorders, information of which may or may not be desired, and
may not require clinical intervention, but it stands as a modern choice over existing technology. In view of the significant
demands in achieving the NBS objectives, NGS is expected to bring both these ends (NBS with modern technology)
together. However, this definitely calls for making changes in the current policy, ethics and legal considerations (Table
1).
∙ NGS-NBS: Whole Genome, Exome or Gene Panel: At the most basic level, before establishment of
NGS-NBS, the first thing to be sorted out is how is NGS to be used in a screening program? NGS being a most
versatile tool can be implemented in different ways to provide information about the entire genome or
only the gene coding region or certain target genes in a selected panel known to be involved in disease
pathogenesis.
Generally exome sequencing has an edge over sequencing of the entire genome, owing to its low cost and easy
interpretation in terms of disease. But the exome covers only 1% of the human genome: thus any DNA variation in the
non-protein coding region will obviously be missed. Furthermore, exome capturing by hybridization can
introduce substantial amount of coverage variability that will have impact on comparative analyses. Also,
copy number and structural variations (CNVs and SVs), as well as some insertions, deletions and block
substitutions are difficult to detect in exome capture data (Belkadi et al., 2015; Meynert et. al., 2014).
These studies highlight the technical upper hand of whole genome sequencing over exome sequencing in
providing an intrinsically richer data of polymorphisms outside the coding region and disclosing genomic
rearrangement. With the steep reduction in the cost of DNA sequencing in recent years (Wetterstrand,
2013; Young S, 2014), the main economic benefit of using exome sequencing is nullified as more sequence
information now could be obtained by using WGS, thereby cost effectively supporting the use of WGS in
NBS. To explore the possibility of establishing WGS-NBS a study was performed recently, which compared
the screening results of 1,696 infants by the state-run NBS program and whole genome sequencing for 27
disorders. Though WGS yielded fewer false positive results as compared to TMS-based NBS, the frequency
of results with uncertain significance was quite high. The conclusion of the study was that WGS might
be used in complementation with the present TMS-based NBS assays (Dale et al., 2015). Conversely, a
recent online survey to analyze the professional opinion of genetic counselors about the use of whole genome
sequencing in the newborn period identified that majority of the respondents felt that presently WGS should
not be used in NBS and if it were to be used, it should not be mandatory. They considered that accurate
interpretation of the result, more extensive consent process, pre and post-test counselling, comparable cost and
turnaround time must be achieved before using NGS in NBS (Ulm et al., 2015). Howard et al. in 2015
have suggested to perform targeted analysis of genes that are clearly involved in a specific disease with
effective and accepted preventive or therapeutic intervention (Howard et al., 2015). However the choice of
panel of genes to be tested will depend upon the epidemiological prevalence which is not uniform across the
world.
∙ Data storage and retrieval: cost and privacy: Sequencing the genome of all the newborns will generate a
huge amount of data and proper data analysis and storage will be required. The cost of the TMS-based NBS procedure
(2011) in the European Union ranges from
€ 0.46 per newborn to
€ 43.24 which is much lower in comparison to the cost
that will be needed for sequencing neonate genome and analyzing the data (Frank et al., 2013). The real budget for the
entire process of screening by applying genomic sequencing is far more than the proposed
$1000 as it does not include the
cost of data analysis, family notification and follow-ups and confirmatory testing (Mardis et al., 2010). On
an average 353,000 babies are born per day around the world and the economic feasibility of sequencing,
analyzing and maintaining the vast amount of data generated is questionable. In the case of late onset
disorders or a fatal disease the information regarding the result of sequencing must be given to the child
after he becomes mature and decides to know his disease status. Fully guaranteeing the governance and
privacy of this information until being disclosed is not possible (Chadwick et al., 2013). However, as the
speed at which technology is progressing, in the near future it is likely that more advanced cost effective
sequencing technology will emerge, thus there might actually be no need to store such information and
sequencing could be done when desired at a later age. But this option will invalidate the much boasted
utility of NGS at an early age and using the information generated for aiding personalized medicine in the
future.
∙ Variants of unknown clinical utility: Incorporation of NGS in NBS will increase the number of uncertain
variants simultaneously increasing the burden on the parents and the care providers (Cooper and Shendure, 2011).
Additionally, it will also lead to an upsurge in the pressure on the laboratory and clinicians to determine the clinical
validity of the variant at the earliest. A combination of variants might be detected in some newborns which may never
lead to occurrence of disease. This will cause consequences of over-treatment in an otherwise healthy child and will result
in unnecessary psychological and financial burden on the family. On the other hand, as sequencing is not totally free from
error and also there are chances that some variations might get missed depending on the sequencing platform used, an
infant may get deprived of early diagnosis and ameliorative or preventive therapy (Knoppers et al., 2013;
Clark et al., 2011). Interpretation of results might also vary among the laboratories and there might exist
discrepancy in assigning a variation as pathogenic or inconsequential hence causing under-diagnosis or
over-diagnosis of disease. Keeping all this in mind it is advisable to determine whether or not to return
uncertain results to the parent.and whether to store the data until validation of these variants and to then
notify it to the parents Also, as the status of variants of unknown significance keeps on changing and is
mostly reclassified over time, it is important to plan follow-up procedures and family notification wisely
without raising anxiety among the parents. The follow up procedure in a country like India will become
even more problematic as most of the families come from remote areas and are often impossible to trace
again.
∙ Unsolicited findings: Although the main emphasis of the NBS program is centered around what is most
beneficial for the child and its expansion to NGS is expected to maximize the benefits to the child’s health, exome or
whole genome sequencing can often reveal probabilistic information about the relatives in the extended family also. The
primary concern of the clinician is to decide on how to return these results to the parents. Though the information of an
adult onset disease may not be required for the child, it might still have clinical implications for the parents or
relatives. It is a conflicting situation for the clinician to decide whether to disclose it to the concerned
at-risk individual as it may hamper the child’s right to an open future. Many attempts have been made to
categorize these unsolicited findings and decide which of these to be disclosed, but not in the screening context
(Bredenoord et al., 2011; Berg et al., 2011). More recently the Public and Professional Policy Committee of
the European Society of Human Genetics, the Human Genome Organization Committee on Ethics, Law
and Society, the PHG Foundation and the P3G International Pediatric Platform have recommended that
unsolicited findings which lead to a preventable or treatable health problem should be communicated (Howard
et al., 2015). Such ethical issues need to be considered and it is advisable to counsel the parents about
such consequences before the test is performed. In case of untreatable diseases, it is recommended that the
information must not be given to the parents but to the child at the proper age after consent (Shannon,
2014).
∙ Need for clinicians trained in genetics: A large number of variants are identified in sequencing the newborn
genome and the clinical relevance of most of them is not so straight forward. Relatively few doctors receive significant
training in genetics and related molecular sciences, and thus lack the background needed to effectively interpret the results
of a genetic test. What to report back to the parents is often a difficult judgment call for these clinicians and if this issue
is not addressed before extending NBS to NGS, it may increase the number of cases of medical malpractice, where a
physician can be held responsible for not being able to detect or disclose the genetic risks preceding the eventual
manifestation of the genetic disorder. Though not in context of newborn screening, but a recent case in Connecticut in
which a woman sued her physician for failing to warn her that her family history of breast cancer also
implied a possible genetic risk for ovarian cancer (Downs v. Trias, 2012) provides some insight into the
bigger picture of NBS upgradation and its pitfalls. One factor that can help reduce liability risks is to
improve the knowledge and training of physicians on genetics-based healthcare. But unluckily, most medical
schools have only recently started training students in genetics, and many physicians feel that they are
not well trained to address genetic issues (Richard et al., 2011).On the other hand, the fear of missing
important genetic information and being held for medical malpractice might force the physician and the
policy makers to return more positive results to the parents for which the follow up results may be normal
but still it can have negative psychological impact on the parents (Hewlett et al., 2006; Johanna et al.,
2012).
∙ Informed consent: NBS is usually conducted without an explicit consent because it is seen to be in the best
interest of the child’s health. However, for genetic screening informed consent is the utmost requirement and the case is
no different in genetic NBS also. The biggest concern in obtaining parental consent is that who should
convey the complicated genetic counselling to the parents and get the informed consent? Are the nurse and
physician well trained for this or a genetic counselor must be appointed for this purpose? Will it be possible to
give such facilities in smaller hospitals and medical centers where most of the babies are born? Would an
information brochure be sufficient for resolving parental queries regarding genetic screening and whether
most of the parents, who have minimal genetic testing experience, can actually understand the complex
genetic information (Harvey, 2014)? A survey was recently carried out on parents’ opinion of whole-genome
sequencing for newborns, if it were offered by newborn screening programs or pediatrician services. In both
scenarios, 70% of parents expressed interest in whole-genome sequencing, citing test accuracy and the ability to
protect a child from developing a disease as important factors in their decision-making process. But rest of
the parents expressed no interest in newborn WGS and were concerned about the “privacy of results”,
“potential for results to be used to discriminate against their child,” and that results could be used for
research (Goldenberg et al., 2014). Thus the major threat for genetic NBS is that some parents might
completely opt out of NBS due to fear that the detection of certain genetic variations in their newborn can
jeopardize obtaining health or life insurance, or even school acceptance and future employment (Landau et al.,
2014). This might have serious consequences for an infant who has a disorder that needs immediate medical
intervention.
5 Testing the Ground Reality of NGS
For implementation of a robust technology like NGS in a mass newborn screening program, the main focus should not be
just technologically biased; it should also be tested for its long and short term impact on the family and the child. The
crucial question here is whether large-scale genomic sequencing can provide useful medical information beyond what
current newborn screening is already providing and at what economical and emotional cost? To address these issues and
to analyze the technical, clinical, practical and ethical aspects of genomics research in the newborn period, the NICHD
(National Institute of Child Health and Human Development) and the NHGRI (National Human Genome Research
Institute), both parts of the NIH (National Institutes of Health) had launched 4 pilot programs in the year 2013
and allotted a fund of $25 million to four grantees over five years. These grantees include: Brigham and
Women’s Hospital, Boston where the genome sequence-based screening for childhood risk and newborn illness
will be studied in both sick and healthy infants by employing whole genome sequencing; Children’s Mercy
Hospital, Kansas city where the researchers are examining the benefits and risks of using rapid genomic
sequencing technology in the NICU population and trying to return the results in 50 hours; University of
California, San Francisco are conducting exome sequencing utilizing newborn blood spots for disorders that are
or are not currently screened for in NBS with an agenda to improve and expand NBS; and University of
North Carolina at Chapel Hill that is performing exome sequencing of healthy infants and infants with
known disorders. These projects are presently ongoing and in the years ahead we may look forward to
finding more realistic answers to the current ambiguity regarding the application of genomic sequencing in
NBS.
6 Conclusion
Implementation of NGS in NBS would require stretching the benefits related to NBS i.e. from what is good for the infant,
to what might be potentially good for the infant, to what might be good for the family (e.g., reproductive benefit or
health benefits for family members), or to what might be beneficial for the society at large (research), thus in a way
diluting the primary goal of the screening program. Presently, in view of the haze surrounding the use of WGS or WES in
NBS, it seems not likely to fit within the available public health-care system due to the practical, financial and ethical
challenges that are making this vision difficult to achieve. Though the outcomes of the pilot projects on large-scale
assessments of the risks and benefits of genome sequencing for newborns will aid in designing the guidelines for NBS
expansion in the near future, as of now the topic of newborn genome sequencing as a public health initiative remains
contentious. It is recommended that such a program could be conducted but as a commercial supplement with
consent.
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