This study highlights both the challenges and opportunities in the application of NGS to clinical diagnosis in patients with intellectual disabilities/congenital anomalies. In cases where we found a clear and likely cause of the condition, this conclusion depended on the knowledge of Mendelian diseases associated with the relevant genes. Two of these genes are already well known: TCF4
; however, the mutations we detected were novel. The example of EFTUD2
is of very particular interest. Before the recent identification of this gene, a possible case could be made on the basis of seeing de novo mutations in two of our patients, although we failed to show a functional effect of one of the two variants in the available tissue. Subsequent comparison of their phenotypes revealed a number of similarities. This example shows that a discovery paradigm focusing on a broad range of conditions provides an important complement to the more common current strategy of combining patients with similar conditions on strictly clinical criteria. By studying the genetics of a broader range of conditions as we did, it is possible to make a careful assessment of any phenotypic overlap of patients that have possible causal mutations in the same genes. In this way, it may be possible to identify conditions with broader phenotypic presentations than is possible in the strictly ‘phenotype first’ framework. However, we do note that confirmation that EFTUD2
is causal required its recent identification by Lines and colleagues.7
It is noteworthy that our study of only 12 patients pointed toward the possibility of EFTUD2 involvement in two of the cases. If a programme such as was used here were applied to many hundreds and eventually thousands of unexplained conditions, it is very plausible that many new genes would be nominated and confirmed using exactly this strategy.
Furthermore, information gained from genome sequencing as described here, focused on a broad range of patients, will likely expand the phenotypic spectrum of many currently well-known genetic disorders. Clinical decisions regarding whether or not to perform a genetic test largely depend on how well the patient fits the clinical description of the disorder. Although mutations in TCF4
are known to cause the well described PHS, the patient in this study did not exhibit two of the most common and differentiating symptoms of this disorder (periods of hyperventilation and seizures), and although the condition was considered, the diagnostic yield was not thought to be high enough to warrant testing. Similarly, the patient with the SMAD4
mutation that is known to cause Myhre syndrome did not show a typical manifestation of this syndrome. It is possible that there are many well described genetic conditions in which the variability in the phenotypic spectrum is not currently appreciated, and NGS may facilitate considerable broadening of this spectrum. The real power of diagnostic sequencing will depend on establishing very large databases that include mutations of interest and corresponding phenotypes. For example, intellectual disabilities and/or congenital abnormalities occur in approximately 3–4% of children,48
and a majority of these are due to underlying genetic causes, yet close to 50% of children with one or both of these phenotypes remain undiagnosed.50
It is likely that a high proportion of these undiagnosed cases will start to be sequenced annually in the next few years, creating the opportunity for very large databases that will permit the identification of currently unrecognised genotype-phenotype connections.
The suggestive finding for NGLY1 is also of particular interest. Rather than being a gene known to be responsible for a Mendelian disease with phenotypic similarity to the patient under study, this gene clearly acts in the same pathway as the known genes causing the Mendelian disorder that had been considered for the child, that is, a congenital disorder of glycosylation. This case illustrates how we can leverage known information about the function of a gene, and in particular its action within a pathway already implicated in Mendelian disease, to help identify new genetic diagnoses.
Our work also demonstrates the importance of the use of ‘general’, non-gene-specific functional evaluation of gene expression to confirm the pathogenicity of a variant. Since the de novo mutation in TCF4 had not been described before and involved a splice site, it made a strong but not definitive case for causality. Functional studies demonstrated that the mutation in TCF4 disrupts splicing and results in a protein targeted for degradation, which confirms causality. This work, therefore, helps to establish a general paradigm for such clinically motivated sequencing which includes not only the identification of candidate variants but also a generalised function evaluation of their impact on gene expression and splicing. However, as the number of sequenced patients increases, and as these data are increasingly shared in public databases, the need for functional work for some variants will decrease as the same variants are shown to occur in multiple patients with similar presentations (as for the SMAD4 variant in trio 3).
It is also important to emphasise that the paradigm we adopted in this study is likely to be similar to how NGS would be applied in clinical genetics practice, since general genetics clinics would have patients with widely differing phenotypes. Our study demonstrates the type of patients that would be sequenced in these clinics and provides data regarding expectations of finding a cause, the importance of functional assays for probable variants and the value of pre-screening patients to determine eligibility for NGS. With our inclusion and exclusion criteria, we set out to maximise the likelihood that an underlying undiagnosed genetic condition was present in each of the enrolled patients, and found causal or likely and interesting variants in 8/12 patients. It is also likely that in clinical practice, partial explanations would be detected for diverse manifestation in the same patient, as in trio 4 in our study, emphasising the complexity of genetic counselling for such a patient whose manifestations are likely to be due to more than one underlying genetic cause. Establishing a diagnosis is often of value even when a clear change in treatment is not indicated by the diagnosis. For example, close and ongoing observation for seizures is now indicated for patient 5 (TCF4
), and avoidance of medications that may trigger seizures, such as antihistamines.52
The family can be informed that the disorder is due to a de novo variant, and in the absence of parental mosaicism, other family members are not at risk, and with future pregnancies the recurrence risk for the parents is low. Additionally, they can learn about PHS, have a better idea of future expectations, and reach out for support from families with similarly affected children. Similarly, patient 11 (SCN2A
) should avoid common anti-epileptic drugs whose primary mechanism is sodium channel inhibition, since these exacerbate symptoms in patients with SCN1A
A confirmed molecular diagnosis may also protect patients from incorrect diagnoses that could lead to unhelpful therapy options.
While cost benefit analyses were not the focus of this work, it is interesting to note that some of the patients who now have a genetic diagnosis, underwent many genetic tests prior to exome sequencing at a considerable estimated cost (eg, more than $22 000 were spent on laboratory investigations in Trio 2) While estimating the real costs of exome sequencing is difficult, it is already clear that in some cases, interrogating genes one by one or in panels will rapidly lead to greater total costs than exome or whole-genome sequencing. While these considerations are encouraging, as is the success rate of six likely genetic diagnoses out of 12 cases (with one further case likely explained partially), this work was performed in a research environment and there will be many challenges involved in a transition to fully clinically based applications. Itemising those challenges, from cost and reimbursement to the type and manner of communication to the families (including the issue of incidental findings), is beyond the scope of this work, but we would highlight two challenges in particular. First, in our experience, laboratory-based functional analysis is an important part of the evaluation, and it remains unclear how this would be incorporated into routine clinical application of NGS, even as NGS is beginning to be offered by commercial laboratories as a clinical test. Second, this work required substantial manual interrogation of both sequence data and candidate genes. Although variant calling procedures are continually improved and there are likely to be routines developed to simplify the process of candidate identification,54
it seems likely that for the foreseeable future, some level of expert judgement will continue to be required to identify causal mutations from sequence data, which will contribute to the cost and time of this type of diagnostics. Currently, it is difficult to imagine how the level of both variant inspection and functional evaluation could be provided as part of routine clinical diagnostic testing. These current essential functions, therefore, present a significant challenge to the use of NGS to provide genetic diagnoses.
Finally, we note that there are a number of reasons that causal variants may have been missed in some trios in this study. One important factor is that we do not have a comprehensive understanding of the function of most genes. For genes whose function is not well characterised, extensive functional follow-up may be required to assign causality to a de novo or homozygous variant carried by an individual patient. We may also fail to detect some causal variants. Exome sequencing does not capture all exons, nor non-coding regulatory regions, and structural genomic variants such as CNVs are difficult to recognise. Additionally, variants within captured regions may be missed by the mapping/variant-calling algorithms. In the future, we anticipate this approach will be improved by the use of whole-genome sequencing and improved variant identification, although for the foreseeable future a small proportion of the genome will remain refractory to high throughout sequencing. It is also possible that causal variant(s) may exert their effects through more complex inheritance patterns than investigated in this study.
In summary, this work indicates that the application of NGS should be strongly considered in all cases where a genetic condition is strongly suspected but traditional clinical genetic testing has proven negative. Furthermore, in some cases at least, it is likely that NGS will prove faster and less expensive than the long diagnostic odyssey many families now endure. However, our work, like that of others, offers the cautionary note that it will probably be possible to identify very strong candidate variants in any sequenced genome and that further studies such as functional assays or multiple patients with mutations in the same gene will often be needed to establish causality. Considerable attention must be paid to establishing appropriate standards of evidence before the results of NGS are used to influence patient care, and establishing such standards will be a major challenge for NGS in the clinic.