Welcome to Strand Genomics-A Monthly E-zine from Strand Life Sciences
Solving the Metabolic Riddle: Genetic Analyses Reveal the Cause Behind Hypoglycaemia and Seizures Evident in a Child
– Dr. Shefali Sabharanjak
Strand Life Sciences
- A 9-year old girl patient presented with hypoglycaemia and seizures, beginning at the age of 1.5 years.
- The patient also presented with vomiting, hepatomegaly, and increased sensorium.
- Genetic testing was advised to understand the causes of these persistent symptoms.
- A mutation in the FBP1 gene was identified, after careful and intensive analysis of patient’s genome.
- An anomalous region was underrepresented in NGS readouts- a feature that was picked up by Strand NGS – Strand’s custom-designed software for analysis.
- Sanger sequencing was provided as an additional validation, free of cost to the patient.
A 9-year-old girl, Arpita, was brought to a prominent hospital in Bangalore for a consultation with a renowned endocrine specialist. Generalized seizures and hypoglycaemia were her principal health issues. In addition to these symptoms, she also suffered from vomiting, hepatomegaly and increased sensorium. Blood tests showed that she had high serum lactate levels as well. Episodes of hypoglycaemia and seizures had started when Arpita was 1.5 years old.
The doctors handling the case advised a liver biopsy as well as genetic tests to understand whether inherited genetic mutations were the cause of Arpita’s health issues.
Family Tree – Pre-Genetic Testing
Arpita’s father’s mother and mother’s father were siblings. Essentially, her parents were cousins and therefore were in a consanguineous marriage. Given this family history, her physician prescribed the Strand Clinical Exome Test to ascertain whether an inborn error of metabolism was present in the child.
Figure 1. Arpita’s Family Tree
Results of the Clinical Exome Test
Arpita was found to be homozygous for a mutation in the FBP1 gene. This gene codes for an enzyme – fructose-1,6-bisphophatase – that is involved in the synthesis of glucose from substances like lactic acid, amino acids and glycerol. Deficiency of this enzyme has been linked with metabolic acidosis, ketosis, elevated levels of serum lactic acid, and even coma (Li et al. 2017).
Figure 2.Identification of a mutation in the FBP1 gene
Since Arpita is homozygous for this mutation, bearing two mutant copies of the gene, she suffers from a complete deficiency of the fructose-1,6-bisphophatase enzyme, manifested as hypoglycaemia and periodic seizures.
Strand’s Bioinformatics Software Enabled Deep Genetic Analysis
In Arpita’s case, an insertion of a 331 base pair ALU sequence in exon 2 of the FBP1 gene was detected after careful analysis. The genomic region of the FBP1 gene was underrepresented in the NGS readouts, compared to readouts from other samples, in the first round of analysis (Figure 3, Double arrowhead). Normally, this underrepresentation would have been missed out in the analytical workflow, owing to reduced number of readouts. However, Strand’s proprietary bioinformatics platforms – Strand NGS and StrandOMICS – have been designed to identify such features of NGS readouts and flag them for further analysis.
Figure 3. NGS analysis of Arpita’s DNA with low readouts of the FBP1 gene (double arrowhead)
In Arpita’s case, this genomic fragment was then analyzed again by Sanger sequencing. Insertion of the ALU sequence in the FBP1 gene was revealed by Sanger sequencing. This insertion was predicted to cause a frameshift mutation, resulting in the formation of an incomplete protein product.
Figure 4. Sanger sequencing of the FBP1 gene led to the identification of an ALU insertion (region between arrowheads) resulting in a loss of function mutation
- The Strand Clinical Exome Test was prescribed to understand the genetic causes of symptoms such as hypoglycaemia and periodic seizures in a 9-year old child.
- Arpita was found to be homozygous for a mutation in the FBP1 gene, caused by an insertion of an ALU sequence in exon 2.
- Identification of the ALU insertion was facilitated by critical analysis enabled by Strand NGS – Strand’s proprietary bioinformatics software – followed by Sanger sequencing offered by Strand at no additional cost to the patient.
- Arpita’s parents were counselled and advised about management strategies such as appropriate food choices for Arpita.
- Mutation-specific testing was advised to the family, especially since Arpita has a sibling. Arpita’s parents are expected to be heterozygous for the same mutation and hence their chances of transmitting two copies of the same mutation to another child are 25%.
Li, N. et al., 2017. Clinical and Molecular Characterization of Patients with Fructose 1,6-Bisphosphatase Deficiency. International journal of molecular sciences, 18(4). Available at: http://www.ncbi.nlm.nih.gov/pubmed/28420223 [Accessed May 23, 2017].
Lung Cancer Snapshot: India
Lung cancer is reckoned amongst the most common cancers, worldwide. Estimates from GLOBOCAN (2012) suggest that 12.9 % of all new cases of cancer are lung cancer cases. In India, incidence of lung cancer cases has risen sharply during the period of 2005-2010, with a continuation of an upward trend in 2011-2014. Adenocarcinoma (30.2%) and Squamous cell carcinomas (44.7%) are the predominant sub-types of lung cancer, in India. Mutations in EGFR, with targeted therapeutics designed to act against specific mutations (del 19 and L858R), are evident in ~40% of adenocarcinoma cases.
(Ref data and images: Dubey, A.K., Gupta, U. & Jain, S., 2016. Epidemiology of lung cancer and approaches for its prediction: a systematic review and analysis. Chinese Journal of Cancer, 35(1), p.71. Available at: http://www.ncbi.nlm.nih.gov/pubmed/27473753 [Accessed June 13, 2017].
Liquid Biopsy – A Convenient Solution To Monitor Cancer Recurrence
– Dr. Shefali Sabharanjak
Strand Life Sciences
- Suneet Varma, a 55-year-old colorectal cancer patient was advised to take Strand’s pan-cancer StrandAdvantage 152-gene est.
- The StrandAdvantage 152-gene cancer test was used to identify a KRASG13D mutation from the solid tumor biopsy as well as from a liquid biopsy (a blood sample from the patient) at the time of initiation of cancer treatment.
- Liquid biopsy tests performed at 4-month and 8-month intervals helped to establish the absence of residual cancer in the patient.
- Strand’s highly sensitive liquid biopsy tests have provided accurate snapshots of cancer progression to the patient.
- Liquid biopsy based follow-up tests may be performed at desired time intervals to monitor progression of cancer in every patient. These tests can circumvent limitations of PET-CT scans such as exposure to radiation and access to scan facilities.
A diagnosis of cancer is not easy to live with. Cancer treatment is a long, drawn-out, expensive and emotionally taxing journey. Quite often, the waiting period between treatment and confirmatory tests like a PET-CT scan is the most anxious period for a patient. Radioactive tests like PET-CTs cannot be performed too often as well. In such cases, a blood based test for ascertaining the presence or absence of residual tumor tissue would be a highly suitable alternative to understand a person’s cancer status. Recent research has shown that liquid biopsy tests can be used effectively to track the presence or absence of cancer DNA and can be used to predict the prognosis of the patient, even before cancer is detected on PET-CT scans (Tie et al. 2016; Zheng et al. 2016; Ai et al. 2016; Pécuchet et al. 2016).
Suneet Varma, a 55-year old bank employee (name and profession changed to protect patient privacy), was diagnosed with colorectal cancer in August 2016. He was an avid badminton player, a bike enthusiast and a father of two young kids, aged 7 and 11 years. The diagnosis of cancer had turned his life completely upside down.
Suneet’s tumor showed signs of invading tissues adjacent to the wall of the colon and hence surgical removal of the tumor was advised by a prominent oncologist in Hyderabad.
In addition to surgery, genetic profiling of the solid tumor as well as follow-up liquid biopsy tests were advised to Suneet. Tumor cells as well as normal cells release their DNA into the bloodstream when they die as part of the natural turnover of cells in various tissues. This DNA, called cell-free DNA (cfDNA) can be harvested from blood and can be checked for presence or absence of genetic mutations that have been identified from the solid tumor as well. The process is akin to assigning a barcode to an item and using that tag to track it.
A biopsy of the solid colorectal tumor as well as a 20ml blood sample (essentially a ‘liquid’ biopsy) from Suneet were sent to Strand Life Sciences for genetic analyses in August 2016.
Results of Genetic Testing
Strand offers a pan-cancer test – the StrandAdvantage 152-Gene Test- that can assay for genes that are frequently mutated in all cancers. This broad-ranging cancer test is useful in identifying genetic mutations that can support the choice of therapeutic options in the form of chemotherapy as well as targeted therapeutics.
In Suneet’s case, this pan-cancer test helped to identify the presence of the KRASG13D mutation in the solid tumor biopsy
Figure1. Genetic Analysis of Solid Tumor
The same mutation was also detected in cell-free DNA (ctDNA) obtained from the blood sample provided by him at the same time.
Figure 2. Liquid Biopsy Test 1 – August 2016
These initial results established the fact that tumor DNA from the colorectal cancer (marked by the identified mutation) was present in Suneet’s blood.
Suneet was prescribed chemotherapy, following the surgery, to target residual cancer, if any.
Keeping Tabs on the Cancer with Liquid Biopsy
Four months after the surgery, Suneet sent another blood sample to Strand Life Sciences for a follow-up liquid biopsy test.
In December 2016, cell-free DNA from his blood was analyzed again and the KRASG13D mutation was NOT identified.
Figure 3. Liquid Biopsy Test 2 – December 2016
This follow-up liquid biopsy test is performed at a high sensitivity of detection. The test can detect the presence of the mutation even if it is present at a concentration of 1 mutant DNA / 1000 normal DNA molecules. Therefore, absence of the mutation in cell-free DNA suggests that tumor DNA (from residual colorectal cancer) is not present in Suneet’s blood. Hence it is safe to conclude that Suneet’s colorectal tumor was removed completely and the chemotherapy has been effective in killing remnant tumor cells.
In cancer parlance, we refer to people as ‘survivors’ or ‘people living with cancer’ instead of ‘people cured of cancer’ and with good reason. One has to make allowances for the fact that a cancer may start growing again, if some cancer cells escape all the given treatment.
In order to understand whether Suneet is still free of cancer, another blood sample was sent to Strand in April 2017.
Figure 4. Liquid Biopsy Test 3 – April 2017
The marker mutation KRASG13D was not detected in cell-free DNA from his most recent blood sample. The absence of this mutation in cell-free DNA indicates that Suneet has remained free of cancer in the past 8 months, post-surgery.
An additional advantage is that the results of the follow-up liquid biopsy tests were obtained within 5 working days.
Figure 5. Summary of Liquid Biopsy Tests For Monitoring Suneet’s Cancer
We have shared these results with Suneet’s oncologist who has been highly appreciative of the accurate results provided by these tests. The ease with which the progression of cancer has been monitored in this case is a significant improvement in cancer care.
- Genetic analysis of a solid colorectal cancer biopsy and a concurrent liquid biopsy sample allowed for identification of a characteristic mutation – KRASG13D – in Suneet Varma, a 55-year-old bank employee.
- Liquid biopsy samples provided by the patient at 4-month intervals were analyzed for the presence of the genetic signature of the tumor.
- The characteristic genetic marker was NOT identified in cell-free DNA isolated from the patient, in two follow-up LB tests, despite using highly-sensitive DNA detection techniques (1:1000 sensitivity), indicating the absence of recurrent cancer. Suneet has been relieved to know that his cancer has been controlled and that it can be detected early, if the cancer recurs.
- Liquid biopsy based personalized genetic analysis of cancer has provided significant emotional relief to a cancer survivor. These tests can be availed of at any desired frequency (monthly, bi-monthly or quarterly) and are free of limitations like exposure to radiation and accessibility to PET-scan facilities.
Ai, B. et al., 2016. Circulating cell-free DNA as a prognostic and predictive biomarker in non-small cell lung cancer. Oncotarget, 7(28), pp.44583–44595. Available at: http://www.ncbi.nlm.nih.gov/pubmed/27323821 [Accessed February 16, 2017].
Pécuchet, N. et al., 2016. Base-Position Error Rate Analysis of Next-Generation Sequencing Applied to Circulating Tumor DNA in Non-Small Cell Lung Cancer: A Prospective Study. M. Ladanyi, ed. PLoS medicine, 13(12), p.e1002199. Available at: http://www.ncbi.nlm.nih.gov/pubmed/28027313 [Accessed April 24, 2017].
Tie, J. et al., 2016. Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer. Science Translational Medicine, 8(346), p.346ra92-346ra92. Available at: http://www.ncbi.nlm.nih.gov/pubmed/27384348 [Accessed February 16, 2017].
Zheng, D. et al., 2016. Plasma EGFR T790M ctDNA status is associated with clinical outcome in advanced NSCLC patients with acquired EGFR-TKI resistance. Scientific reports, 6, p.20913. Available at: http://www.ncbi.nlm.nih.gov/pubmed/26867973 [Accessed February 16, 2017].