Genetic variation causes or contributes to the pathophysiology of all neurologic disease. Recent technologic advances have enabled better understanding of how genes influence disease and opened the door to revolutionary improvements in diagnosis, prognostication, and treatment across medical disciplines. Although the importance of genetics in neurologic disease has been known for decades, the high cost of genetic analysis and limited ability to treat many neurologic disorders, once diagnosed, kept neurogenetics out of most clinical practices. Tremendous advances in DNA sequencing and the development of new tools to correct human gene mutations, however, have brought genetic analysis and gene therapy to the cutting edge of clinical neurology. These advances herald a new era of personalized medicine in pediatric and adult neurology. Here, I discuss these recent advances and how they are changing the way we practice neurology.

The human genome contains approximately 3 billion nucleotides, fewer than 2% of which form genes that encode proteins that are the building blocks of cells.1 The other approximately 98% of the human genome does not encode proteins and is thought to regulate expression of nearby genes. Variation in protein-coding genes and noncoding regulatory DNA can significantly modify disease risk, with some gene variants dramatically increasing risk and others reducing it. The combination of an individual’s coding and noncoding genomic variants define her or his risk for a given disease (eg, genetic penetrance and expressivity). Genetic disorders caused by single gene mutations with high penetrance are considered monogenic (eg, spinomuscular atrophy [SMA] or Rett syndrome), whereas diseases caused by mutations that must occur together across multiple genes are considered polygenic (eg, ischemic stroke or migraine).2 Monogenic diseases are subcategorized into dominant or recessive depending on whether 1 or 2 copies of a mutant gene must be present to cause disease.

DNA Sequencing

The first human genome draft sequence took over a dozen labs, $3.0 billion, and more than a decade (1990-2001) to sequence.3 Now, an individual can have his or her entire genome sequenced commercially in a day for less than $1,000.4 Although many advances have come together to enable this vast growth in DNA-sequencing potential, among the most important is the advent of next-generation sequencing (NGS).5 Earlier methods sequenced 1 region of DNA at a time; NGS generates millions to billions of distinct sequences in parallel, exponentially increasing sequencing speed, accuracy, and ease of analysis. Indeed, individuals can now independently send cheek swabs or blood samples to commercial laboratories that will sequence their entire genomes for a fee, opening the door to using a person’s genome to guide diagnosis, prognosis, and management of their condition.

Gene Editing

The wealth of knowledge generated by DNA sequencing advances has led to new insights into numerous diseases. However, until 5 years ago, genetic mutations that cause neurologic disease had been viewed as an immutable problem because gene editing technology was largely limited to research and difficult to scale clinically. Basic research in bacteria identified a primitive adaptive immune system that uses the Cas9 protein and clustered regulatory interspaced short palindromic repeat (CRISPR) sequences to guard against invading viruses. This system is remarkably flexible and can be engineered to edit nearly any genomic region, paving the way for researchers to adapt this system to edit the genome of human cells.6-8 Huge advances have been made in this young field of gene editing over the past 5 years to optimize efficiency for use in humans.9 There are now dozens of companies using CRISPR/Cas9 to develop new gene therapies, the first of which has just begun clinical trials in people with Leber’s congenital amaurosis.9

Gene Therapies

Once it has been determined how a genetic mutation leads to a disease pathophysiology, there are a number of tractable approaches for delivering gene therapy. The primary mission of gene therapy is to deliver genetic material to selectively replace, repair, or control the expression of a mutant gene in the cell type(s) involved in the respective disease. This is a particularly difficult problem for neurology because of the vast heterogeneity of nervous system cell types as well as the difficulty of penetrating the blood-brain barrier. The 2 major approaches for delivering gene therapy are nonviral and viral.10 Both approaches have had tremendous success, and multiple additional clinical trials are in progress (Table). Among the most promising nonviral gene therapy approaches, antisense oligonucleotides (ASOs) are short nucleotide sequences, chemically modified to enter cells without degradation.11 Chemical modification of ASOs is an active area of research and can dramatically affect bioavailability as well as provide tissue specificity, in some cases.12 Typically, ASOs are designed to target and degrade mutant RNA or to promote alternative splicing, the latter of which is exemplified by nusinersen, approved by the Food and Drug Administration (FDA) to treat SMA. There is also great interest in using modified liposomes—artificial membrane-bound droplets—to deliver genetic material to cells after fusion with the cell membrane.13 Viral-mediated gene therapy is particularly versatile because viruses can express full-length genes and are thus amenable for gene replacement. Viruses also have tropism for specific tissues and cell types that can be leveraged to develop more targeted therapies. Adeno-associated virus (AAV), already endemic in humans without known adverse effects, is the most common type of virus engineered for gene therapy.14 The AAV-based gene replacement therapy for a type of Leber’s congenital amaurosis was approved by the FDA in 2017, AAV-based onasemnogene gene replacement therapy was approved for SMA in 2019, and trials are underway for mucopolysaccharidosis, Batten’s disease, and multiple other genetic conditions.

Emerging Applications of DNA Sequencing

Sequencing DNA is already an important part of diagnostic workup in pediatric neurology for identifying known causal gene mutations (eg, MECP2 mutations in Rett syndrome) and for discovering novel disease-causing mutations.15 As more is learned about causal relationships between genotypes and clinical presentations, there is increasing interest in including NGS as part of newborn screening. A recent pilot effort using dried blood spots as samples has been largely successful,16 raising important clinical and ethical questions about how to implement this technology responsibly in common practice. Phenylketonuria (PKU) demonstrates the clinical importance of early detection and intervention for certain metabolic disorders, and as gene therapies improve, early gene replacement or correction will be equally important. There are justified concerns, however, that whole genome sequencing can lead to a slippery slope of genetic intervention for DNA variants with small contributions to disease risk but unknown benefits in the context of the individual’s genetic profile. Given the early stage of gene therapy, it is prudent to be cautious with this powerful intervention until the long-term risks are better understood.

Adult neurology is also beginning to see the fruits of DNA sequencing. As in pediatric neurology, DNA sequencing is aiding discovery and diagnosis of mutations that result in adult-onset neurologic disease (eg, Charcot-Marie-Tooth disease, or cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy [CADASIL]). Additionally, NGS has also become an important way to identify novel viruses and microbes in cerebrospinal fluid (CSF) from individuals with subacute encephalitis.17 In some cases, the ability to rapidly sequence and interpret samples can have profound effects on clinical management. For example, in people who are septic, sequencing blood samples can rapidly identify the microbial source as well as potential antimicrobial resistance genes to guide appropriate treatment.18 In persons with gliomas, DNA analysis is being used for prognostication,19 and in the rapidly emerging field of oncogenomics, tumor genome sequences can be used to guide choice of chemotherapy. These sequencing methods are slowly moving from research laboratories and out-of-pocket fee-for-service commercial services into more mainstream clinical practice (Figure).

Figure. Clinical Applications of Next-Generation DNA Sequencing.

Click to view larger

Figure. Clinical Applications of Next-Generation DNA Sequencing.

There is also exciting potential for the rapidly expanding pool of genomic information from the hundreds of thousands of people who have had their genomes analyzed. These data are being used to perform genome-wide association studies (GWAS) that link genomic variation with specific diseases.15 Among the most promising is the GWAS for coronary artery disease (CAD), in which individuals’ sets of genomic variants were used to accurately predict their risk for CAD.20 This approach identified that 8% of the population had a threefold higher risk than the other 92% and demonstrates that a simple genetic screening test could quickly identify which people need aggressive early intervention. Similar predictive success is observed for atrial fibrillation, type 2 diabetes, inflammatory bowel disease, and breast cancer.20 As additional genomes of carefully phenotyped patients are sequenced, the genome-disease associations learned from GWAS can be extended to additional diseases and even used to identify more homogenous patient populations for clinical trials.

Applied Gene Therapy in Neurology

As discussed, advances in gene therapy have already reached neurology practice. The most notable is nusinersen for treatment of SMA, a neuromuscular disease characterized by progressive muscle wasting, respiratory failure, and death. Basic research identified mutations in SMN1, which encodes survival of motor neuron protein 1, as the cause of SMA.21 A related gene discovered at the same time, SMN2 has overlapping function with SMN1, but the messenger RNA (mRNA) produced is unstable and expressed at low levels in motor neurons. Stability of SMN2 mRNA can be improved with the ASO that directs RNA splicing machinery to include exon 7 of SMN2 to provide more stable mRNA and thus more SMN2 protein.22,23 A limitation of nusinersen, however, is its administration requires quarterly intrathecal infusions. As a result, there has been a major push toward viral-based gene replacement approaches that will function for years after a single dose, which has led to the recently FDA-approved AAV-9 based gene replacement therapy for SMA.24 It should be noted that in the pivotal AAV gene-therapy study for SMA, systemic delivery of AAV significantly elevated liver enzymes in several patients. An additional limitation of AAV-based gene therapy is that up to a quarter of people already have neutralizing antibodies against AAV by adulthood, although this is much lower in the pediatric population.25 Despite these limitations, the tremendous success using AAV for gene replacement in SMA has prompted considerable interest in treating a host of other neurologic diseases including Rett syndrome, Duchenne muscular dystrophy, Charcot–Marie–Tooth disease, and more. Pediatric neurologists are beginning to see these advances in practice and there is great promise for adult neurologists on the horizon with clinical trials underway using gene therapy for Alzheimer’s disease, Parkinson’s disease, and antineutrophil cytophilic autoantibody (ANCA) vasculitis.

Future Directions

The future of gene-based diagnosis and treatment is extraordinarily bright. As the cost of DNA sequencing continues to fall, it will greatly expand access to testing and facilitate the discovery of rare disease-causing mutations that previously have been difficult to identify. As genomic information continues to grow, so too will the statistical power needed to accurately correlate genotype and risk for a disease. The lower cost of sequencing is likely to complement and, in some cases, replace current clinical laboratory studies for diagnosing infectious diseases, cancer, and other disorders. As the era of gene therapy has arrived, there continues to be exciting progress in the development of organ and even cell-type-specific viral vectors that will only affect the intended tissues or cells. It is hoped this will dramatically reduce liver toxicity and other side effects of systemic virus delivery. Advances in single-cell genomics are enabling new insights into differences between brain cell types within an individual that should further catalyze more targeted therapeutics.26,27 Extraordinary advances are also being made in the specificity of CRISPR-based gene editing technologies, which are rapidly enabling the translation of this bacterial immune system from bacteria to bedside.


It cannot be overstated how advances in DNA sequencing and gene editing have already and will continue to revolutionize medicine. The successful application of these technologies to diagnose and now treat SMA and Leber’s congenital amaurosis are serving to accelerate similar progress on multiple other neurological and nonneurologic conditions. As DNA sequencing and gene therapies become more common in our practice and provide unprecedented treatments for previously incurable diseases, it is important for us to remember that it was our curiosity about the bacterial immune system that built the tools for human gene editing, our curiosity about viral genetics that enabled AAV-based gene therapy, and our curiosity about RNA splicing that gave us nusinersen to treat SMA. We must continue to invest heavily in these basic sciences in order to build the treatments of tomorrow.

1. International Human Genome Sequencing C. Finishing the euchromatic sequence of the human genome. Nature. 2004;431(7011):931-945.

2. Vgontzas A, Renthal W, Introduction to neurogenetics. Am J Med. 2019;132(2):142-152.

3. Lander ES, Linton LM, Birren B, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409(6822): 860-921.

4. Hayden EC. Technology: the $1,000 genome. Nature. 2014;507(7492):294-295.

5. Shendure J, Ji H. Next-generation DNA sequencing. Nat Biotechnol. 2008;26(10):1135-1145.

6. Cong L, Ran FA, Cox D, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339(6121):819-823.

7. Jinek M, Chylinski K, Fonfara I,et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816-821.

8. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823-826.

9. Tsai SQ, Joung JK. Defining and improving the genome-wide specificities of CRISPR-Cas9 nucleases. Nat Rev Genet. 2016;17(5):300-312.

10. Puhl DL, D’Amato DR, Gilbert RJ. Challenges of gene delivery to the central nervous system and the growing use of biomaterial vectors. Brain Res Bull. 2019;

11. Rinaldi C, Wood MJA. Antisense oligonucleotides: the next frontier for treatment of neurological disorders. Nat Rev Neurol. 2018;14(1):9-21.

12. Lokugamage MP, Sago CD, Dahlman JE. Testing thousands of nanoparticles in vivo using DNA barcodes. Curr Opin Biomed Eng. 2018;7:1-8.

13. Sharma G, et al. Cell penetrating peptide tethered biligand liposomes for delivery to brain in vivo: biodistribution and transfection. J Control Release. 2013;167(1):1-10.

14. Hudry E, Vandenberghe LH. Therapeutic AAV gene transfer to the nervous system: a clinical reality. Neuron. 2019;101(5):839-862.

15. Lappalainen, T., et al., Genomic analysis in the age of human genome sequencing. Cell. 2019;177(1):70-84.

16. Barben J, Modgil A, Layek B, et al. Retrospective analysis of stored dried blood spots from children with cystic fibrosis and matched controls to assess the performance of a proposed newborn screening protocol in Switzerland. J Cyst Fibros. 2012;11(4):332-6.

17. Kennedy PGE, Quan PL, Lipkin WI. Viral encephalitis of unknown cause: current perspective and recent advances. Viruses. 2017;9(6).

18. Sinha M, Strunk T, Jiang P, et al. Emerging technologies for molecular diagnosis of sepsis. Clin Microbiol Rev. 2018;31(2).

19. Yan H, Parsons DW, Jin G, et al, IDH1 and IDH2 mutations in gliomas. N Engl J Med. 2009;360(8):765-773.

20. Khera AV, Chaffin M, Aragam KG, et al. Genome-wide polygenic scores for common diseases identify individuals with risk equivalent to monogenic mutations. Nat Genet. 2018;50(9):1219-1224.

21. Lefebvre S, Bürglen L, Reboullet S, et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell. 1995;80(1):155-165.

22. Groen EJN, Talbot K, Gillingwater TH. Advances in therapy for spinal muscular atrophy: promises and challenges. Nat Rev Neurol. 2018;14(4):214-224.

23. Skordis LA, Dunckley MG, Yue B, Eperon IC, Muntoni F. Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts. Proc Natl Acad Sci U S A. 2003;100(7):4114-4119.

24. Mendell JR, Al-Zaidy S, Shell R, et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med. 2017;377(18):1713-1722.

25. Harrington EA, Sloan JL, Manoli I, et al. Neutralizing antibodies against adeno-associated viral capsids in patients with mut methylmalonic acidemia. Hum Gene Ther. 2016;27(5):345-353.

26. Renthal W, Boxer LD, Hrvatin S, et al. Characterization of human mosaic Rett syndrome brain tissue by single-nucleus RNA sequencing. Nat Neurosci. 2018;21(12):1670-1679.

27. Tanay A, Regev A. Scaling single-cell genomics from phenomenology to mechanism. Nature. 2017;541(7637):331-338.

WR receives unrestricted investigator-initiated research grants from Teva and Amgen.