It was like the Wild West when next generation sequencing entered the genomics landscape a dozen years ago, and Avni Santani, PhD, jumped into the excitement as a postdoctoral fellow at Children’s Hospital of Philadelphia. On the horizon, she saw the transformative power of the technology that now enables scientists to interrogate entire genomes in a span of hours or days. Reining in the full potential of these advancements to improve clinical care is a vital part of Dr. Santani’s new role as director of Clinical Laboratories, Strategic Partnerships and Innovation at the Center for Applied Genomics (CAG) at CHOP.
“When I read the publication on the first next generation sequencing instrument, I thought, ‘Wow, this is going to change our world.’” Dr. Santani said. “But I could not have predicted how disruptive this technology has been for the genomics community. For years, in the clinical laboratory, we were used to standardized protocols using sequencing technology like Sanger that were honed and optimized for 20 plus years. There was practically no R&D back then for a molecular genetics laboratory. Now, we have state-of-the-art instruments and technology, but they are in a constant state of improvement, and the informatics tools to analyze and interpret vast amounts of data are still being developed or optimized.”
For the last decade, the genomics community collaboratively has been figuring out which technologies were working, and how can they can get the data they produce to do what they want it to do. Today, they have within sight an affordable whole genome test. As next generation sequencing has quickly become more sophisticated, faster, and less expensive, CAG has been blazing the trail toward applying this genomic technology paired with reliable informatics to facilitate rare pediatric disease diagnosis, prevention, and management.
“This is a pivotal time to be involved in clinical genomics,” Dr. Santani said. “We face new challenges every day, yet at the same time we are making more gene discoveries than ever before and identifying novel treatments for our patients.”
For example, CHOP researchers and CAG analysts recently identified a loss-of-function mutation in the KIF15 gene that is associated with Braddock-Carey Syndrome, a rare disease characterized by craniofacial malformations and low levels of platelets in the blood. In another international multi-center collaboration, CAG led a study team that used whole-exome sequencing to identify variants in the TBCK gene that occurred in 13 individuals (from nine unrelated families) who all had intellectual disability and decreased muscle tone; many also had seizures and changes in the white matter of their brains.
Detecting these disease-causing glitches helps families arrive at a definitive diagnosis, often after pursuing many years of physician visits and medical testing. Knowing the underlying genetic problems can inform their future medical care and help them to understand if other family members are at risk of developing the same condition.
Achieving CLIA Certification
In order to perform clinical grade sequencing and genotyping on patient samples and use these insights to better inform healthcare choices, CAG needed to meet Clinical Laboratory Improvement Amendments (CLIA) certification requirements mandated by the Centers for Medicare & Medicaid Services. That was Dr. Santani’s first hurdle to cross when she accepted her new position with CAG. Dr. Santani also is a director in the division of Genomics Diagnostics in the department of Pathology at CHOP and is an assistant professor of Clinical Pathology at the Perelman School of Medicine at the University of Pennsylvania.
CLIA oversight of laboratories and personnel has a strong emphasis on developing a quality management program — quality control, quality assurance, and quality improvement practices. The goal of the certification process and required bi-annual proficiency testing is to make certain that a laboratory team carefully considers the integrity and accuracy of complex lab procedures; ensures the sensitivity, specificity, and reproducibility of their data; and promotes the safety of patients and research participants. In short, they must continuously adhere to high standards in all aspects of their work and be able to prove it every step of the way.
Dr. Santani and CAG’s staff developed a rigorous and systematic program to continuously monitor quality for CAG’s pediatrics genomics biobank, which includes samples from more than 400,000 people, and all sequencing and genotyping activities. After a detailed onsite inspection from the U.S. Department of Health, CAG received CLIA certification in December 2016.
“This reflects a major accomplishment from the entire team at CAG who worked tirelessly for many months to achieve this milestone,” Dr. Santani said. “The CAG team is excellent and dedicated to the pursuit of quality in every step of the pathway from specimen collection to data analysis.”
A Perfect Match for Collaboration
Hakon Hakonarson, MD, PhD, director of CAG and professor of Pediatrics at Penn, couldn’t agree more. With CLIA certification under its belt along with more than a decade of experience in biobanking and in the generation and management of genomic data, CAG is in a unique position as the only CLIA-certified high throughput sequencing and SNP-array facility that operates within a world-renowned pediatric research institution that has such integrative assets, Dr. Hakonarson said. CAG has the ability to process and deliver results on several thousand specimens a month with the infrastructure and secure cloud-based storage it has in place to handle the vast amount of data that is collected.
“All of what we do is focused on how we can make our discoveries work toward better diagnostics and improved therapies that we can bring to the pediatric clinic,” Dr. Hakonarson said. “CLIA status opens up numerous opportunities for a big research lab like ours to accomplish this.”
Partners and collaborators within CHOP and in other academic, biotechnology, and pharmaceutical organizations worldwide can rely on CAG to support large clinical research studies and clinical trials. Suppose a pharmaceutical company would like to conduct a biomarker trial. The pharma team could choose to establish an entire laboratory and data processing center on their own, which would be an intensive undertaking. Or they could work with CAG’s specialists who already have established the appropriate technical and regulatory framework to streamline these type of projects, all of which are backed up by CHOP’s biobanking efforts.
CAG extended one of those successful genomics collaborations in February with Aevi Genomics Medicine. The company has access to certain data from the CAG biobank to pinpoint genetic mutations underlying specific rare and orphan diseases, and it plans to utilize this information to identify development candidates for advancement into therapeutic and diagnostic products for sick children, according to a press release announcing terms of the agreement.
‘Our Ultimate Goal is to Improve the Health of Our Patients’
CAG also offers an individualized, “one stop” approach for the CHOP-Penn research community. They’ve teamed up with over 60 internal investigators in various ways, such as providing input on grant applications or helping them to generate data through collaboration and access to the CAG biobank for their studies. CAG experts often sit down with study teams to determine the best ways to design, optimize, and validate assays that will help them arrive at the answers they’re looking for — and just as importantly — have confidence when results come back negative, Dr. Santani said.
“The question that most people don’t ask is: What happens when you sequence a whole genome, and you don’t find a pathogenic variant?” Dr. Santani said. “Because analysis of genome data is so complex, there is always the underlying concern about quality and how well was the analysis performed. This is a challenge for the entire genomics community. The inter-disciplinary team at CAG is very experienced both in generation of sequencing data and also in the analysis and management of data. We are familiar with the complexities of our informatics infrastructure, which has been established to meet high quality standards. So, when the results on a whole genome analysis are negative, one can be assured that we have leveraged the strengths of the available informatics tools, and no stone has been left unturned in looking for that pathogenic variant.”
The CAG team applies this same rugged determination as it explores new methods to genome sequencing and analysis. They have created several tools that are implemented by research and clinical laboratories throughout the world. One is an algorithm (Penn CNV) that scientists use to analyze copy number changes that are detected in the genome. That platform has received more than 1,200 citations reflecting its use in the community. Also, genome annotation software developed at CAG (ANNOVAR) helps to map genes and their functions in the coding and non-coding regions of a genome. It has more than 3,000 citations.
“Sequencing technology and informatics tools are changing at a blindingly rapid pace, and clinical researchers are unable to keep up, let alone make informed decisions to improve patient care,” said Dr. Santani, who holds board certifications in Clinical Molecular Genetics and Clinical Cytogenetics. “Our team of 70-plus scientists at CAG are at the perfect nexus to use their expertise to evaluate and develop new technologies, collaborate with researchers and biotech companies, establish the clinical utility of genomic information, and advance medicine. It’s an intellectually rich environment where the focus is on resolving the critical challenges that genomics presents to us almost on a daily basis. Our ultimate goal is to improve the health of our patients by diagnosing patients earlier than we can do today and to also find new treatments to better manage these conditions.”
New Frontiers as Genomics Goes Mainstream
One of those challenges involves coupling genomic data with patients’ electronic medical records (EMRs). CAG is part of a consortium called the Electronic Medical Records and Genomics (eMERGE) network that is now in its third phase. One of the project’s aims, Dr. Hakonarson explained, is to identify and validate actionable genetic variants that impact patients’ health, such as genetic variations that may cause individuals to have adverse reactions to certain drugs, and then integrate that information into EMRs. An overarching question that the investigators are trying to answer is how patients’ knowledge of their genomic results generated by eMERGE and other CAG-related efforts affect their medical, ethical, and financial decision-making.
Genetic carrier screening is one example of how genomics has arrived at the intersection of mainstream clinical applications. Finding variants can be very informative for couples who are at childbearing age, Dr. Hakonarson said. Perhaps they are parents of a child with a rare pediatric genetic disorder and want to find out their risk of conceiving another child with the same disease. Or maybe a couple is planning to start a family and already know that one partner’s ancestral background predisposes them to harboring a certain genetic condition; they may want to confirm if the other partner has the exact same gene variant that could increase the chance that their baby would be affected.
“A child has six billion base pairs — three billion from the father and three billion from the mother that reside in the pair of 23 chromosomes each of our cells harbors,” Dr. Hakonarson said. “Among the millions of base pairs that vary among individuals, one variant may be totally critical to whether a child is healthy or has a major medical condition. It’s incredible. Our CAG analysts are focused on finding that one pathogenic variant after looking at billions of base pairs.”
As interest grows from a consumer standpoint for genetic carrier screening services and the costs become less prohibitive, Dr. Hakonarson anticipates that CAG’s new CLIA status will attract future third-party opportunities, in which CAG would perform the complicated genomic work of isolating the DNA, processing the sequencing data and annotating the information based on all of the variants generated. CAG would provide the third party requesting the information for their clients about the results, and then genetic counselors could help interpret the findings, convey them to their concerned families, and help them understand their genetic risks.
“And we will always ask for the opportunity to use the samples and information we gather in a de-identified way for research,” Dr. Hakonarson said. “This will allow for our biobank to grow much faster and allow for far more productive research to be done, a win-win situation for all.”
As genomics research moves closer to clinical application, the CAG team is ready to tackle these new frontiers to discover and resolve the underlying genetic causes of children’s most complex and extremely rare diseases.