Issue 07 | July 2017
Media Open Day at Strand’s NGS Laboratories
DNA Analysis Provides Accurate Diagnosis of A Birth Defect
In the News
South Asians prone to genetic diseases: CCMB study
Ribosomal DNA Copy Numbers Decrease in Some Cancers
Liquid biopsies provide more information about disease course in non-invasive manner
Call us now to discuss your test requirements!
Welcome to Strand Genomics-A Monthly E-zine from Strand Life Sciences
Strand Life Sciences welcomes you to Strand Genomics, our monthly E-zine that includes articles of interest to physicians. This e-zine brings the latest news in the world of genetic diagnostics, to your doorstep. We present carefully crafted articles as well as curated news in the field of cancer therapy and genetic analyses to support the implementation of personalized medical care. We invite you to peruse as well as share these articles. Please also feel free to write back to us with comments and questions at firstname.lastname@example.org
Do BRCA Mutations Cause Cancers Other Than Hereditary Breast and Ovarian cancers?
Dr. Shefali Sabharanjak
Strand Life Sciences
Mutations in BRCA genes – BRCA1 and BRCA2 – can increase a person’s risk for developing breast and ovarian cancer (Effery S Truewing et al. 1997; Petrucelli et al. 1993). Angelina Jolie is a classic example. Her grandmother died of ovarian cancer, her mother suffered from breast as well as ovarian cancer and her maternal aunt died of breast cancer. When she had herself tested, a pathogenic variant of the BRCA1 gene was identified in her genome. We all know this story. Angelina Jolie opted for risk-reduction mastectomy and risk-reduction salpingo-oophorectomy surgeries to protect herself. She also sparked a debate, worldwide, on ad hoc genetic testing – to be done or not to be done!
The role of the BRCA genes in causing breast and ovarian cancer has been studied in various population groups and across various countries as well (Esra Manguoǧlu et al. 2003; Mannan et al. 2016; Hirasawa et al. 2014; Vaclová et al. 2016). A quick summary of some studies was also published in an earlier issue of this e-zine. So, now the question arises, should BRCA testing be advised only to women? Are men in the clear?
Not really. Cases of male breast cancer, resulting from inheritance of a mutant BRCA gene HAVE been documented (data from Strand’s analysis of some male breast cancer cases, (Benjamin & Riker 2015). The role of BRCA germline mutations in the development of multiple types of cancers has been debated in several studies (van Asperen et al. 2005; Lorenzo Bermejo & Hemminki 2004). The role of BRCA mutations in increasing the risk for cancers at other sites has also been examined in a recent study (Digennaro et al. 2017). Observations from this study suggest that surveillance of BRCA mutations should be extended to men and women alike.
Incidence of Other Cancers in Families Prone to Hereditary Breast and Ovarian Cancer
A group of researchers in Italy have published a study last month, describing their observations from 136 families that were counseled for incidence of HBOC (Digennaro et al. 2017). During genetic counseling, a family tree is constructed to understand whether many members of a family have been diagnosed with cancer or not. These researchers determined the pedigree of each family in this study including family members up to three generations.
The families were then classified into two branches:
- ‘H’ branch where a clear inheritance of HBOC was noted in the family history
- ‘nH’ branch where breast and ovarian cancer were not hereditary
A closer look at the various characteristics of the families in each branch revealed that except for the incidence of HBOC, both branches are well-matched in other characteristics.
Table 1: Cohort Characteristics of Hereditary and Non-Hereditary Categories of Breast and Ovarian Cancer (Digennaro et al. 2017)
|Characteristics||Hereditary Branch||Non-Hereditary Branch||P value (chi-square)|
|Mean Age (years)||65.9||69.1|
|Number of cancers||376||90||<0.0001|
|Breast cancer (number)||182||13||<0.0001|
|Mean Age at Diagnosis||54.4||63.8|
|Ovarian cancer (number)||33||2||<0.0001|
|Mean Age at Diagnosis||52.1||59.5|
|Other Cancers (number)||161||75||<0.0001|
|Mean Age at Diagnosis||65.2||65.4|
Evidently, the incidence of breast and ovarian cancers is higher in the H branch of the study than the nH branch (p < 0.0001). Barring this expected difference, the cohort characteristics of the H and nH branches are similar.
Diving deeper, these researchers also noticed that the overall incidence of lung, kidney, colon, and prostate cancers in the H branch was higher than that in the nH branch.
Table 2: Incidence of other cancers in H (n = 1156) nH (n = 1062) branches of families with hereditary ovarian/breast cancer syndrome (Digennaro et al. 2017)
|Cancer site||Number of cancers in branches||P valuea||Cancer site||Number of cancers in branches||P valuea|
aChi square with Yates correction; bnot significant
The presence of inherited BRCA mutations in the H branch, was also confirmed by genetic analyses.
Table 3. Probability of Suffering from Various Cancers- A Logistic Regression Analysis (Digennaro et al. 2017)
|Neoplasia||Independent variable||Relative Risk||95% CI||P value|
The results of this study show that presence of BRCA mutations in the family bloodline can increase the risk of developing lung, kidney, liver, and larynx cancers, in addition to breast and ovarian cancer.
Since the other cancers are not partial to any gender, the risk of developing these cancers is equal for men and women both. In fact, risk calculations performed by these scientists show that the higher risk for lung, liver, and larynx cancer were independent of gender AND age in the H branch. Women in the H branch had a lower risk for development of larynx cancer when compared with women in the nH cohort; however, gender was not evident as a risk reducing factor for other kinds of cancer.
Similar results were reported in an earlier study as well (van Asperen et al. 2005). Higher risks for developing pancreatic, prostate, bone, and pharynx cancer were observed in relatives of BRCA2 mutation carriers. In order to eliminate bias, relatives were selected as BRCA2 mutation carriers based on pedigree analyses of 139 families, rather than relying on results from genetic testing (van Asperen et al. 2005).
Clearly, these studies show that surveillance of people for the presence of BRCA mutations should be extended to men and women as well. In fact, if a patient has been identified as a carrier of a BRCA mutation, it should serve as a red flag for the entire family, across three generations.
van Asperen, C.J. et al., 2005. Cancer risks in BRCA2 families: estimates for sites other than breast and ovary. Journal of medical genetics, 42(9), pp.711–9. Available at: https://www.ncbi.nlm.nih.gov/pubmed/16141007 [Accessed July 11, 2017].
Benjamin, M.A. & Riker, A.I., 2015. A Case of Male Breast Cancer with a BRCA Gene Mutation. The Ochsner journal, 15(4), pp.448–51. Available at: https://www.ncbi.nlm.nih.gov/pubmed/26730232 [Accessed July 10, 2017].
Digennaro, M. et al., 2017. Hereditary and non-hereditary branches of family eligible for BRCA test: cancers in other sites. Hereditary cancer in clinical practice, 15, p.7. Available at: https://www.ncbi.nlm.nih.gov/pubmed/28559958 [Accessed June 15, 2017].
Effery S Truewing, J.P. et al., 1997. THE RISK OF CANCER ASSOCIATED WITH SPECIFIC MUTATIONS OF BRCA1 AND BRCA2 AMONG ASHKENAZI JEWS Background Carriers of germ-line mutations in. The New England Journal of Medicine, 336(15), pp.1401–8. Available at: https://www.nejm.org/doi/pdf/10.1056/NEJM199705153362001 [Accessed July 7, 2017].
Esra Manguoǧlu, A. et al., 2003. Germline mutations in the BRCA1 and BRCA2 genes in Turkish breast/ovarian cancer patients. Human Mutation, 21(4), pp.444–445. Available at: https://www.ncbi.nlm.nih.gov/pubmed/12655560 [Accessed February 22, 2017].
Hirasawa, A. et al., 2014. Family History and BRCA1/BRCA2 Status Among Japanese Ovarian Cancer Patients and Occult Cancer in a BRCA1 Mutant Case. Japanese Journal of Clinical Oncology, 44(1), pp.49–56. Available at: https://www.ncbi.nlm.nih.gov/pubmed/24218521 [Accessed February 22, 2017].
Lorenzo Bermejo, J. & Hemminki, K., 2004. Risk of cancer at sites other than the breast in Swedish families eligible for BRCA1 or BRCA2 mutation testing. Annals of Oncology, 15(12), pp.1834–1841. Available at: https://www.ncbi.nlm.nih.gov/pubmed/15550590 [Accessed July 11, 2017].
Mannan, A.U. et al., 2016. Detection of high frequency of mutations in a breast and/or ovarian cancer cohort: implications of embracing a multi-gene panel in molecular diagnosis in India. Journal of Human Genetics, 61(6), pp.515–22. Available at: https://www.ncbi.nlm.nih.gov/pubmed/26911350.
Petrucelli, N., Daly, M.B. & Pal, T., 1993. BRCA1- and BRCA2-Associated Hereditary Breast and Ovarian Cancer, Available at: https://www.ncbi.nlm.nih.gov/pubmed/20301425 [Accessed May 24, 2017].
Vaclová, T. et al., 2016. Germline missense pathogenic variants in the BRCA1 BRCT domain, p.Gly1706Glu and p.Ala1708Glu, increase cellular sensitivity to PARP inhibitor olaparib by a dominant negative effect. Human molecular genetics, p.ddw343. Available at: https://academic.oup.com/hmg/article-lookup/doi/10.1093/hmg/ddw343 [Accessed March 22, 2017].
Genomics Think Tank
Conversations with Dr. Gurdeep Sethi
Dr. Gurdeep Sethi is the Founder of Millennium Cancer Center, Gurgaon, India. Dr. Sethi brings with him more than 26 years of expertise in cancer care and disease management including palliative care, pain management and patient advocacy. He is an active member of the American College of Physicians, American Society of Clinical Oncology, American Society of Hematology, Indian American Cancer Association, Texas Medical Association, Harris County Medical Association, Punjab Medical Council, Delhi Medical Council and Thai Medical Council. He has also served as the Director of Medical Oncology for Pan International Oncology cancer centers in India for the last 16 months after having practiced for over 26 years in the United States of America.
Dr. Sethi has shared his views on the clinical utility and implementation of genetic analyses in cancer care.
- How do you see genomics and genetic analyses playing a role in cancer therapy, in India?
Genomics and genetic analysis is the most effective way to manage cancer not only in India, but also across the globe. This is already being routinely implemented in the U.S. Cracking the human genome code was a gigantic task that took over 10 years, with billions of dollars spent on it. It is our duty to make it useful for everyone. Currently hereditary cancers are seen in approximately 10% of the population. These are cancers that occur in patients less than 50 years age group. We see a very strong shift in the trend of an increased incidence of cancer in the younger population in India. I truly believe that there is a greater prevalence of hereditary cancers in this country as compared to the rest of world. The section of the population that is at risk for hereditary cancer will certainly benefit from germline genetic analysis. Ad hoc genetic testing, done before the actual incidence of cancer can help to increase surveillance. This is expected to lead to early detection of cancers as well as personalized treatment.
- Dr. Sethi, In your clinical practice, how has your experience with targeted therapeutics been? Do patients experience a better quality of life with targeted therapeutics than with generic chemotherapy?
With genetic testing we can understand the genetic profile of the cancer. This knowledge leads to choice of targeted drugs that are designed to counter the cellular functions of the mutant proteins. Many patients are already on active chemotherapy sessions. These patients are then reassured in their minds that they DO have alternative directed treatments, if they do not respond to the current treatment regimen. Some patients do shift to the targeted therapy right away due to intolerance to the side effects of chemotherapy. Overall, in the long run I do see targeted therapeutics replacing generic chemotherapy.
- Genetic analyses can provide suggestions for off-label uses of targeted therapeutics, based on the identification of genes that they are approved for, although in a different tissue. This is intended to extend therapeutic benefit to patients. What are your views on this matter?
I do believe that there is a great potential for this. However, due to our basic training, we have been conditioned to prescribe medication that has been clinically confirmed to provide therapeutic benefit, as judged from randomized clinical trials. Cancer medicine however is evolving every day. There is much research and data to process and we still do not understand the full potential of genetic analysis and the benefits of targeted therapy based on the gene mutation. In such a scenario, if there is a targeted protein identified with effective medication available, then I would volunteer to treat the patient based on any literature available after they’ve failed conventional treatment options. The process will be explained to the patient in detail and they will be given the choice.
- What has been your experience in terms of savings of time and cost to a patient, between targeted and generic chemotherapy?
Chemotherapy has been used in oncology for 50 years. New therapeutic drugs are being developed every year. The side effects are better controlled. Patients are managed better and stay out of the hospital most of the time. The cost of chemotherapy has gone down drastically, over time. Targeted therapy has fewer side effects and lesser collateral damage to normal tissue. The cost, however, is a significant deterrent. It is almost 4 to 5 times the cost of current chemotherapy regimens. As time progresses and these medications become generic, they would replace chemotherapy in the long run.
- Dr. Sethi, in your opinion, is a genetic analysis of the tumor essential for therapy choices at the stage of initial diagnosis of cancer or does it work well throughout the course of cancer treatment as well?
Genetic analysis of a tumor can support the choice of therapy at the initial stages of diagnosis as well as throughout the course of cancer treatment. This is absolutely true!! Cancer cells can mutate (change) and present as a disease in multiple variant forms. This essentially means that we need a fresh ‘snapshot’ of the genetic profile of each patient’s cancer, at every stage of the disease. New target proteins are produced due to these mutations. In order to introduce new drugs to target these changes, an understanding of the genetic profile of a cancer – quite like time-lapse photography to capture a sequence of events – is absolutely vital. Liquid biopsy is precisely the technique to use, in order to achieve this. Follow-up liquid biopsy is now becoming a norm as soon as resistance to any current chemotherapy or disease progression are noted.
- Liquid biopsy has the potential to provide personalized, longitudinal cancer care to each and every patient. Do you foresee any hurdles or challenges in the inclusion of this new technique for monitoring the progression of cancer, in the Indian scenario?
The challenge I see in this setting is getting the patient to understand the concept in the first place and then accepting increasing cost with the recurring tests. This process of longitudinal cancer care with follow-up liquid biopsies is already a normal practice in the United States. This is because it makes sense, scientifically, and insurance companies cover the cost. In the long run, I agree that liquid biopsy-based personalized cancer therapy will become the norm in India as well.
Elusive Large Genetic Structural Variants Causing Rare Diseases Meet Their Match in Strand’s Clinical Exome Test
– Dr. Shefali Sabharanjak
Strand Life Sciences
Limb Girdle Muscular Dystrophy, GNE myopathies, Marinesco-Sjogren Syndrome, Ectodermal dysplasia, hypophosphatemic tumoral calcinosis, Joubert syndrome – this is a small representative list of rare disorders that have been confirmed using Strand’s Clinical Exome Test for developmental disorders and inborn errors of metabolism.
Rare disorders, as the name suggests, have a low incidence rate, and consequently, the opportunities to understand them thoroughly, are also few and far between.
Strand offers a Clinical Exome Test that has been used for confirming the incidence of several rare developmental disorders as well as inborn errors of metabolism (single genes mutated). This NGS test covers more than 4500 genes and has been used in the diagnoses of 1500 cases so far.
Diagnosis of Joubert Syndrome
An interesting case was recently referred to Strand Life Sciences, recently. Snigdha (baby of Madhulika, names of patient and mother changed to protect privacy) showed signs of slow development at the age of 5 months. She had poor eye contact with people around her and her head nodded persistently, displaying lack of control over neck muscles. A brain MRI was obtained to understand the reasons for delayed developmental milestones. The MRI showed the presence of a classic molar tooth sign in the cerebellar region, a clear indication of Joubert Syndrome (Brancati et al. 2010). The consulting physician advised the child’s parents to send a blood sample for genetic analysis. The Strand Neurodevelopmental Disorders test- a sub-panel of the Strand Clinical Exome Test- was prescribed for baby Snigdha. This test is designed to ascertain mutations in 19 genes associated with Joubert syndrome.
Table 1. Results of Genetic Testing For Joubert Syndrome
In this case, baby Snigdha was found to be homozygous for a deletion of the NPHP1 gene.
Under normal circumstances, deletions of genomic regions are not detected in NGS analyses. However, Strand’s proprietary bioinformatics software – Strand NGS – has been designed to identify anomalous readouts, as well as copy number variations (See Figure 2, readouts between yellow arrowheads) that can potentially flag insertions and deletions. The software flagged a complete absence of both the copies of the NPHP1 gene. The neighboring genes have been read accurately with a copy number of 2. This feature of Strand NGS – Strand’s custom-designed software for analysis of NGS readouts – has been instrumental in identifying insertions and deletions in several other cases as well. Homozygosity was confirmed using PCR methods as well, as an additional support for accurate diagnosis.
Figure 1. Identification of total absence of copies of the NPHP1 gene (area between yellow arrowheads)
As can be seen in the image, gene readouts corresponding to the NPHP1 gene were completely missing (black bars). In contrast, readouts from the adjacent genes (green and teal bars) indicate that two copies of those genes are present in this patient.
Detection of Large Genomic Insertions
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 hypoglycemia 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 hypoglycemia and seizures had started when Arpita was 1.5 years old.
Arpita’s 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.
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).
Strand’s Bioinformatics Software Enabled Exhaustive 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.
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. 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.
A detailed description of this particular case was published in the previous issue of Strand Genomics.
Performance of Strand’s Clinical Exome Test
Strand’s Clinical Exome panel has been instrumental in providing accurate analyses of genetic abnormalities including large insertions and deletions. The detection rate of pathogenic mutations with the Strand Clinical Exome Test is 50 %, in a collective analysis of several hundred cases. Detection of pathogenic mutations by Whole Exome Sequencing NGS tests from leading diagnostic providers is usually between 24-34% (Lee et al. 2014; Stavropoulos et al. 2016; Valencia et al. 2015).
Several labs provide NGS analyses of Whole Exomes of target tissues. However, the coverage of each gene in whole exome sequencing is 20X or less. This results in inefficient detection of variants, a problem that we have discussed in detail in an earlier issue of Strand Genomics. In contrast, the clinical actionability and depth of coverage of Strand’s Clinical Exome Panel are superior to Whole Exome Sequencing tests. In fact, the depth of coverage (essentially the number of times we ‘read’ a particular sentence) is higher than the recommended threshold provided by Illumina for the Clinical Exome Test.
Brancati, F., Dallapiccola, B. & Valente, E.M., 2010. Joubert Syndrome and related disorders. Orphanet Journal of Rare Diseases, 5. Available at: https://www.ojrd.com/content/5/1/20 [Accessed May 22, 2017].
Lee, H. et al., 2014. Clinical Exome Sequencing for Genetic Identification of Rare Mendelian Disorders. JAMA, 312(18), p.1880. Available at: https://jama.jamanetwork.com/article.aspx?doi=10.1001/jama.2014.14604 [Accessed July 17, 2017].
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: https://www.ncbi.nlm.nih.gov/pubmed/28420223 [Accessed May 23, 2017].
Stavropoulos, D.J. et al., 2016. Whole-genome sequencing expands diagnostic utility and improves clinical management in paediatric medicine. Available at: https://www.nature.com/articles/npjgenmed201512.pdf [Accessed July 17, 2017].
Valencia, C.A. et al., 2015. Clinical Impact and Cost-Effectiveness of Whole Exome Sequencing as a Diagnostic Tool: A Pediatric Center’s Experience. Frontiers in pediatrics, 3, p.67. Available at: https://www.ncbi.nlm.nih.gov/pubmed/26284228 [Accessed March 21, 2017].Show full article.