Customer Interview

Using NGS to Study Rare Undiagnosed Genetic Disease

Whole-exome and transcriptome sequencing prove beneficial in uncovering mutations and pathways associated with rare disease.

Using NGS to Study Rare Undiagnosed Genetic Disease

Interview: Using NGS to Study Rare Undiagnosed Genetic Disease


A rare disease affects only a small group of individuals. Yet, the term small is relative. In the United States, a rare disease is defined as affecting 200,000 people or fewer, according to the National Center for Advancing Translational Sciences.1 In Europe, a disease is considered rare when it affects fewer than 1 in 2000 individuals.2 In Korea, the Korean Undiagnosed Disease Program (KUDP) has determined that a condition is rare when it affects fewer than 20,000 people or when the prevalence is unknown due to its rarity.3 All these small groups add up. With as many as 7000 known rare diseases, and many yet to be discovered, tens of millions of people all over the world are affected. Unfortunately, many of them are likely to go undiagnosed and untreated due to a lack of reliable and accurate clinical tools. Murim Choi, PhD is working to change the future for some of these patients.

Dr. Choi, Associate Professor at the Seoul National University (SNU) College of Medicine, works closely with local clinicians to study pediatric neurodevelopmental disorders. As a Principal Investigator in the SNU College of Medicine Functional Genomics Lab and a contributor to the KUDP’s numerous research efforts, Dr. Choi’s goal is to understand the molecular mechanisms of various human diseases using next-generation sequencing (NGS) and other forms of genetic analysis. He is a pioneer in using whole-exome sequencing (WES) to identify genetic variants that might play a causal role in different rare disease conditions. Early in his career, he taught himself the Perl programming language to enhance his ability to understand the data that his studies produce. Ultimately, Dr. Choi’s goal is to contribute to the diagnosis of rare disease patients who often present with puzzling symptoms.

iCommunity spoke with Dr. Choi about his love of Perl programming, his use of WES over the last decade, and how NGS will be used in the future for rare disease research.

Murim Choi, PhD is an Associate Professor at SNU, a Principal Investigator in the SNU College of Medicine Functional Genomics Lab, and a KUDP research contributor.

Q: What first sparked your interest in a career in genetics research?

Murim Choi (MC): I was originally trained as a developmental biologist, receiving a Bachelor’s degree in Molecular Biology. I always wanted to learn about heart development and ended up at Duke University studying how the heart developed in the mouse system. It wasn’t genomics, more embryology and mouse genetics. In early 2000, I was studying phenotypic variation due to a genetic variant that was causing heart problems in a specific mouse strain. It was a project that led me into the world of genetics and the genotype–phenotype relationship. I decided that I wanted to study human genetics.

Q: When did you begin studying the genetics of human disease?

MC: In 2007, I started working in Dr. Richard Lifton’s lab at Yale University. We were using Illumina SNP arrays to look at genotype, heterozygosity, and copy number variation (CNV) on disease genomes. We were also performing genome-wide association studies (GWAS). It was a whole new world to me.

"Today, the cost of NGS has dropped dramatically and we perform WES routinely as one the first steps in assessing for rare genetic disease."

Q: What prompted you to teach yourself the Perl coding language?

MC: I decided I needed to study coding so that I could manage the data that we were producing in Dr. Lifton’s lab. I went to the bookstore with my one-year-old son and picked up a book on Perl, which was the main tool for bioinformatics then. During the day, I was in the lab preparing DNA and performing GWAS. After I put my son to bed at night, I studied Perl. I really enjoyed it.

There were no public tools for GWAS data analysis, so I used my knowledge of Perl to design the genomic alteration and downstream analysis. I showed Dr. Lifton the GWAS data of the cohort that I had been working on and he liked the analysis. He said that I shouldn’t go back to the lab bench and recommended that I had the skill to analyze and manage the genetic data, which I agreed with. It felt quite powerful to be able to digest and make sense of the data.

Q: Dr. Lifton’s lab was known for its work on cardiovascular diseases. How did you become involved in rare and undiagnosed genetic disease research?

MC: My work in Dr. Lifton’s lab allowed me to look at an individual’s genome in a very broad way. He was kind enough to provide me with all the data sets he had for analysis. When the first NGS systems were introduced in 2009, Dr. Lifton asked me to set up the WES pipeline in the lab. One of the first projects was using NGS to exome sequence a patient who had been diagnosed with a rare disease.

Q: Why are some of these rare diseases so difficult to diagnose?

MC: Rare diseases are mostly Mendelian and monogenic. Their symptoms can be diverse and complex, with onset early in development. These features make it hard for clinicians to diagnose. Therefore, where the patient lives and the experience of the clinician play a significant role in diagnosing rare disease. If it is an easy and straightforward rare disease, the patient is diagnosed instantly in a clinic close to their home. However, not all physicians have enough experience diagnosing rare diseases, raising the possibility that they might misdiagnose the patient. In South Korea, local pediatricians might refer those patients to clinicians at the SNU Children’s Hospital. I’m affiliated with the hospital, which is right across the street from my lab. My lab is situated in a great location to study these diseases.

"...our group is performing more functional genomic techniques, including CRISPR and single cell–based NGS analyses."

Q: What can genetic assessments offer pediatric patients with rare disease?

MC: Even with a genetic assessment, we don’t always have a treatment available for patients with rare disease. If it’s a de novo mutation, we can let the parents know that subsequent children won’t likely to be afflicted by the disease. If the disease is the result of recessive variants, we can advise the parents to be prescreened for the mutation before having a second child.

Sometimes, we can provide direct help to the child and the family. One scenario is when the identified mutations are in the enzyme of a metabolic pathway, resulting in the lack of an important metabolite. In those cases, we can supplement those metabolites to ameliorate the symptoms.

Another scenario is when we identify variants where there are current drugs available that target those mutations for non-rare diseaes. For example, several years ago there were two cases where we identified patients who harbored de novo mutations in genes critical for autoimmune and hyperinflammatory responses, respectively. A functionally mimicking antibody to enhance defective gene function alleviated the severe autoimmune systems in one patient.4 A signaling inhibitor to reduce increased gene function relieved vasculopathy in the other patient.5

However, most of the patients that we analyze are pediatric neurology patients and their cases are complex. Neurological disease is where breakthroughs in understanding the underlying genetics are most needed.

Q: What are the advantages of NGS techniques over older genetic technologies in identifying rare disease–associated variants?

MC: When I first joined the Lifton lab, I was a young geneticist and ignorant about what NGS could offer. I heard people talking about how sequencing was getting more robust and I wasn’t sure what that meant. The only sequencing that I knew of was Sanger sequencing and I couldn’t understand the advantage of making it faster.

I soon realized how much faster NGS was and that it provided the best coverage of the genome. Yet, at SNU, when I first collaborated with the clinicians, we were careful not to use NGS immediately because of its cost. We would start with Sanger or focused screening approaches based on clinical interpretations. If they didn’t provide the answer, we’d move to WES because it provided more coverage for unknown genes and could identify mutations in unexpected genes, providing explanations for novel syndromes and unrecognized diseases.

Today, the cost of NGS has dropped dramatically and we perform WES routinely as one the first steps in assessing for rare genetic disease. It is no longer cost prohibitive to use NGS to resequence every rare disease patient to obtain a better understanding of the genetics of their conditions. It enables us to stratify patients by genotypes that were unrecognized previously. I think we’ll see the use of NGS increase for rare disease studies.

"Illumina sequencing data has gotten better and better over the last 10 years. We have a quality control pipeline, but it rarely raises a red flag because the data are very stable."

Q: How does NGS enable your own research?

MC: We perform two kinds of NGS studies in our laboratory; patient genome sequencing, such as whole-genome sequencing (WGS) and WES, and functional genomics. Both approaches make good use of NGS techniques. Functional genomics studies assess the impact of variants of unknown significance on cellular function. We can use that information as we follow rare disease patients over time. It provides us with a very comprehensive reading.

Q: What inspired you to begin using WES in your studies?

MC: We used WES in Dr. Lifton’s lab to analyze six subject samples, all without any known mutations and five from patients conceived from consanguineous marriages. At the time, it was much easier to detect homozygous variants in those individuals. One of the five patients had been diagnosed with Bartter syndrome. We didn’t find any of the mutations associated with Bartter syndrome, but did identify a mutation in the chloride channel gene. We discussed the finding with a postdoc who had insight into the biology of Bartter Syndrome in renal disease. She suggested that the cause of the patient’s symptoms was due to disruption of the chloride channel associated with congenital chloride diarrhea rather than Bartter syndrome. Fortunately, there’s a drug to treat congenital chloride diarrhea and it alleviated some of the subject’s symptoms. It was the first study to show how NGS techniques could inform diagnosis in rare disease patients.6

Q: How has your use of WES evolved in your research?

MC: It’s been 10 years since we reported that WES could be used to help with the diagnosis of rare disease. We’ve used WES to identify TONSL mutations in SPONASTRIME dysplasia,7 GLBI mutations in GM1 gangliosidosis,8 and GABBR2 mutations in Rett syndrome.9 The value of WES isn’t a secret anymore and it's now moving into routine use in applied industry and clinics. The pediatric clinical laboratory that we collaborate with now performs WES routinely. When they find something interesting, they bring it to us to study in greater depth.

WES is a very stable technique. However, as basic scientists, we feel that WES should be covered by the clinical labs. That’s why our group is performing more functional genomic techniques, including CRISPR (clustered regularly interspaced short palindromic repeats)—based screening methods and and single cell–based NGS analyses. While our methods may have changed, our objectives have not. We are still interested in understanding phenotype–genotype relationships. We’re using our functional genomics data to make sense of the patient phenotypic data to identify the presence of rare disease.

"I think we’ll see more clinical labs performing WES on rare disease patient samples. There is also a place for WGS in the clinic."

Q: What is the focus of the SNU School of Medicine Functional Genomics Lab?

MC: Our lab is focused on the genetics of ultrarare disease. We have a pool of cohorts and have made some interesting discoveries of novel genotype–phenotype correlations. We are using the information to establish collaborations using GeneMatcher, a platform to share gene and phenotype information with clinicians and researchers worldwide. It’s given us a window into the biology of what’s really going on in the cell, enabling us to see if the novel gene we’ve identified could be a potential therapeutic target.

We are also performing data analysis studies. We have accumulated approximately 700 probands and their parents’ data. While it is a small study compared to the large data projects currently underway in the United Kingdom and by major consortium, we can use the data for controls and comparisons as we build the database.

As I stated earlier, we don’t have consanguineous marriages or many homozygous variants in Korea. However, we do have a high occurrence of compound heterozygous variant patients. We believe that if we can increase the number of proband families in our database, we’ll have the information necessary to create a gene panel for use in prescreening potential parents for rare disease and associated mutations. As NGS becomes less expensive, the cost of the test will become more affordable and available to couples thinking of having children.

Q: What NGS sequencing systems do you use?

MC: We used the HiSeq 2000 System when we set up our WES pipeline in Dr. Lifton’s lab. In our Functional Genomics Lab, we outsource the sequencing to a service lab that uses HiSeq and NovaSeq 6000 Systems. Illumina sequencing data has gotten better and better over the last 10 years. We have a quality control pipeline, but it rarely raises a red flag because the data are very stable.

Q: How does your research team contribute to the KUDP?

MC: Dr. Jong-Hee Chae is a pediatric clinician, a principal investigator at the KUDP, and one of my closest collaborators. She runs the KUDP pediatric neurology program and has created a database of 5000 neurodevelopment cohorts. Recently, we successfully transplanted our WES pipeline into her lab. I’m involved in the genetic interpretation of the different variants found and participate in clinician meetings to review the analysis.

Q: What do you see as the future of NGS in studying rare and undiagnosed genetic diseases?

MC: I think the use of NGS methods will continue to grow in the future. There will be more of a distinction between the methods clinical laboratories perform and the methods used in basic science labs.

I think we’ll see more clinical labs performing WES on rare disease patient samples. There is also a place for WGS in the clinic. Linkage analysis studies can be performed with WGS to narrow down the region being investigated. RNA-Seq of tissue samples can also be used to pin down the gene of interest. Ultimately, I believe that WGS will be used routinely in Korean clinical labs.

In basic science labs studying rare disease, we’ll continue to perform RNA-Seq if we have a solid hypothesis of what we are looking for in the genome. We’ll also run more functional genomics screening experiments using CRISPR and other synthetic primers so that we can eventually compare our discoveries with the phenotype information from patients.

Learn more about the products and systems mentioned in this article:

NovaSeq 6000 System

  1. National Center for Advancing Translation Sciences. Rare Disease Day at NIH 2019. Accessed October 11, 2019.
  2. Eurordis, Rare Disease Europe. About Rare Diseases. Accessed October 11, 2019.
  3. Kim SY, Lim BC, Lee JS, et al. The Korean undiagnosed diseases program: lessons from a one-year pilot project. Orphanet J Rare Dis. 2019;14:68.
  4. Lee S, Moon JS, Lee CR, et al. Abatacept alleviates severe autoimmune symptoms in a patient carrying a de novo variant in CTLA-4. J Allergy Clin Immunol. 2016;137:327–330.
  5. Seo J, Kang JA, Suh DI, et al. Tofacitinib relieves symptoms of stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy caused by 2 de novo variants in TMEM173. J Allergy Clin Immunol. 2017; 139:1396–1399.
  6. Choi M, Scholl UI, Ji W, et al. Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. Proc Natl Acad Sci USA. 2009; 106:19096–19101.
  7. Chang HR, Cho SY, Lee JH, et al. Hypomorphic Mutations in TONSL Cause SPONASTRIME Dysplasia. Am J Hum Genet. 2019 Mar 7;104(3):439–453. doi: 10.1016/j.ajhg.2019.01.009. Epub 2019 Feb 14.
  8. Lee JS, Choi JM, Lee M, et al. Diagnostic challenge for the rare lysosomal storage disease: Late infantile GM1 gangliosidosis. Brain Dev. 2018 May; 40(5):383–390. doi: 10.1016/j.braindev.2018.01.009. Epub 2018 Feb 10.
  9. Yoo Y, Jung J, Lee YN, et al. GABBR2 mutations determine phenotype in Rett syndrome and epileptic encephalopathy. Ann Neurol. 2017 Sep; 82(3):466–478. doi: 10.1002/ana.25032.