From April 2018 to January 2019, 10 cases were enrolled into RapidSeq, ranging from 3 days to 4.5 months old. Seven were patients from NICU, 2 were patients from CICU, and 1 was a stored fetal DNA sample where the mother was 12 weeks pregnant with a second pregnancy. Nine of the 10 patients were of Chinese ethnicity, while 1 was of Filipino and Japanese descent (Table 1).
In 4 of the 10 cases (40%) a genetic diagnosis was achieved (Table 4), with one further case diagnosed on re-analysis of data 2 years later. For 3 of the 4 cases, the diagnosis led to changes being implemented in the patient’s management plan. The median turn-around time (TAT) for results was 9.5 working days (range 5-19 days) (Table 2).
Diagnoses and clinical actionability
For cases 2, 6 and 10, the discovery of an underlying unifying diagnosis provided more clarity to the patient’s care team, allowing more targeted management and surveillance for underlying complications. For example, case 6 had a de novo known pathogenic mutation C>G in the SAMD9 gene, consistent with a diagnosis of MIRAGE (Myelodysplasia, Infection, Restriction of growth, Adrenal hypoplasia, Genital phenotypes and enteropathy) syndrome, which is consistent with the patient’s clinical presentation. Prior to diagnosis, the child was being managed by multiple specialists, including the neonatologist, endocrinologist, and haematologist, with multiple investigations done to assess for the etiology of her underlying manifestations. With identification of her diagnosis, this allowed for a more holistic and targeted management of her underlying condition, and furthermore also facilitated surveillance for other complications, such as for haematological malignancy and immunodeficiency.
RapidSeq is resource-intensive, requiring the support of a multidisciplinary team including the intensive care physician, medical subspecialists, clinical geneticists, GCs, laboratory scientists, and bioinformaticians simultaneously. Facilitating RapidSeq requires effective coordination between the clinical and laboratory team, and workflows were refined throughout the study period to optimize timely processing of the results. Factors contributing to prolonged turnaround time included difficulties with pre-test counselling, sample limitations, and bioinformatics processing.
Pretest genetic counselling
In critically ill patients, parents are often overwhelmed by the ongoing medical issues and status of the child, experiencing feelings of anxiety, anger, depression and increased stress. This can sometimes impede pre-test counselling, affecting the ability of parents to process the multitude of information presented to them and hence give informed consent. Furthermore, given the absence of genetic non-discrimination regulations in Singapore, concerns about a genetic diagnosis affecting insurance is commonly a barrier to testing locally, often delaying initiation of testing until insurance concerns are sorted out. These can hamper the use of genomic sequencing as a diagnostic tool for these patients, given the concerns for secondary findings, which has been noted at 1.6% in our local cohort. For the purposes of this pilot, we chose to only report primary findings and not incidental or secondary findings. Interestingly, in our cohort, none had a delay in initiating testing due to insurance concerns, possibly due to the urgency of diagnosis taking precedence over concerns about insurance implications in a critically ill patient.
Availability of appropriate sample for DNA extraction
Sample collection can also be delayed due to acute medical events in the patient, such as the need for blood transfusions taking priority to sample collection, hence alternative means should be sought. For case 6, the patient had received a packed cell and platelet transfusion 3 and 9 days prior to recruitment. In our institution, it is recommended to wait for 2 weeks following the last transfusion before collection of blood sample to prevent leukocyte contamination. As such, the patient underwent a skin biopsy for skin fibroblast culture and subsequent DNA extraction, adding an additional 11 days before DNA was ready for analysis.
Managing parental and primary physician expectations
Although obtaining a genetic diagnosis often has implications in the management of a critically ill patient, parents, and even primary physicians, can sometimes have raised expectations of the test and its impact on the child’s management and prognosis, placing unrealistic expectations on the outcome of the test. It is important to manage parental (and primary physician) expectations of possible result outcomes, which can range from clear cut diagnoses, to variants of uncertain significance, incidental findings, or even a negative result. Case 9 was a preterm infant born at 34+4 weeks with multiple congenital anomalies including tracheo-esophageal fistula, anorectal malformation, left congenital talipes equinovarus, right multicystic dysplastic kidney and low-lying cord. He underwent RapidSeq at day 3 of life which returned negative; this initially made it difficult for parents to understand the extent of his medical condition, as they had hoped that with continued intervention, he would go on to be a normal child. It was then important to communicate to them that in spite of a negative genetic test, the child still had multiple congenital abnormalities that would require long term follow up and medical management.
The median TAT from sample receipt to provisional results in our study was 9.5 days, with a range of 5-19 days. All patients except case 6 had a TAT <14 working days, with the reason for delay being due to the increased time required for DNA extraction from skin fibroblasts.
Cost of testing
At present, we calculate the cost of RapidSeq to be SGD$6000 (USD$4500). Processes will need to continue to be improved to lower costs to reduce the barriers to testing and keep testing sustainable and feasible.
In view of cybersecurity concerns, computers in the Singapore public healthcare sector do not have internet access. Cloud transfer of genomics data to the bioinformatician team at a different physical location is thus not possible, and the genomics data has to be transferred via a physical hard disk that requires effective coordination between the laboratory and the bioinformatics team. The large amount of data generated from the RapidSeq also requires specific informatics needs and tools that are in accordance with clinical testing standard for data management, storage, analysis and archiving. We are exploring alternative options of a cloud-based system that is compatible to the intranet across different public healthcare institutions in Singapore to allow for the continuous provision of data transfer that is necessary in RapidSeq.
Limitations of testing
Bioinformatics filtering process
There are also limits to RapidSeq, whereby only the clinical exome, targeting known genes at that point in time was targeted, or limits with the bioinformatics filtering process. Case 7 was a term infant with multiple congenital anomalies including complex ventriculomegaly with Dandy-Walker malformation, left-sided congenital diaphragmatic hernia, retinal coloboma, and dysmorphic features and was recruited at day 10 of life. However, results returned negative, with parents continuing to hope for active treatment for as long as genetic testing was inconclusive or did not show a lethal genetic condition. She went on to have whole-exome sequencing which found 2 variants of uncertain significant in WDR81, which was associated with congenital hydrocephalus type 3. These 2 variants were initially picked up on RapidSeq, but subsequently filtered out, possibly as the variants were of uncertain significance and, furthermore, only some of the child's features could be explained by it. The child stayed in hospital from birth till initial discharge at 10 months old, and underwent multiple operations and procedures, of which her clinical course was complicated by multiple infections. After discharge, she continued to have multiple readmissions before her subsequent demise at 15 months old.
Lack of local reference genomic databases
Our ability to accurately classify variants in our cohort is also challenged by the fact that Asians are under-represented in population and clinical variant databases (e.g. gnomAD and Clinvar), which has resulted in patients of Asian ancestry being more likely to receive ambiguous genetic test results or variants of uncertain significance. A recent local study of patients with suspected undiagnosed genetic conditions has shown that up to 61% of the variants seen in this multiethnic Asian population are novel. We used trio-based sequencing as a way to improve our variant filtering process as illustrated by reduced number of candidate variants in Table 3.
Evolution of clinical phenotype and need for reanalysis
Furthermore, correlation with clinical phenotype may also be difficult, especially in the paediatric setting, whereby clinical features may develop with age and be less recognizable when young, which has been reported in previous studies. This is seen in our cohort with case 8, a full-term neonate who presented with recurrent apneas, and for whom RapidSeq returned negative; the child was started on anti-epileptic medications, with a presumptive diagnosis of neonatal seizures and remained well. Two years later, the child was noted to have disproportionate short stature, and re-analysis of RapidSeq data showed a pathogenic variant in the FGFR3 gene, consistent with a diagnosis of hypochondroplasia. This variant had been found in the initial analysis but decision had been made not to report it as clinically the child did not have any features of hypochondroplasia, and incidental findings were chosen not to be reported for this research. This emphasizes the importance of re-analysing data from unsolved cases at a later timepoint, which could uncover the presence of variants in newly discovered genes or new phenotype-genotype associations[30-33].