Abstract
Suspecting a genetic etiology for movement disorders of childhood often requires a high index of suspicion due to the heterogenous phenotypic expression, variable penetrance, and the influence of epigenetic modifiers that are largely unknown.
Introduction
Considering a genetic etiology for movement disorders of childhood often requires a high index of suspicion due to the heterogenous phenotypic expression, variable penetrance, and the influence of epigenetic modifiers that are largely unknown. It is, therefore, important to emphasize that neither a period of normality (normal development prior to onset of symptoms) or an association with infectious processes or single static insults, for example, exclude a genetic disorder. Molecular genetic testing has emerged as a powerful approach to identify molecular causes for previously “idiopathic” conditions such as cerebral palsy, dystonia, and parkinsonism. Molecular genetic testing can be done on any DNA-containing specimen (e.g. saliva, skin, or other organ biopsy) and its reliability is less influenced by sampling procedures, storage conditions, and transferring media of the sample under investigation compared to metabolic/biochemical testing of urine, plasma, or cerebrospinal fluid (CSF). In many cases, molecular testing can provide a more accurate diagnosis than biochemical testing. For example, in glucose transporter type 1 deficiency syndrome, a normal plasma to CSF glucose ratio should not preclude molecular testing in the appropriate phenotypic setting.
First-generation gene sequencing (Sanger sequencing) was among the first molecular techniques to be integrated in clinical practice. In this approach, DNA fragments are tagged with labeled nucleotides that are then separated by gel electrophoresis. Then, a chromatogram with the appropriate nucleotide order is generated via laser-emitted light. Sanger sequencing can identify changes or variants in a single gene. It often requires a-priori knowledge of the gene to target based on the clinical and radiological differential diagnosis. Indication-specific gene panels provide a good sequencing “coverage” of genes of interest. This approach can be cost-effective if the target gene is identified correctly. However, due to variable penetrance and allelic heterogeneity in metabolic movement disorders, the diagnostic yield of Sanger sequencing can be limited and time-consuming.
In contrast to Sanger sequencing, next-generation sequencing (NGS) enables a high-throughput parallel sequencing of the entire exome (whole-exome sequencing) or genome (whole-genome sequencing) and the analysis of large amount of data using bioinformatics [1, 2].
The integration of NGS into clinical practice requires appropriate infrastructure and the expertise to analyze and interpret genomic data and provide results in a timely manner. The workflow of NGS includes library preparation, cluster amplification, sequencing by synthesis, and alignment with data analysis (Illumina NGS). The goal of the first step (library preparation) is adding i5/i7 sequences to DNA, which allow for flow cell interaction. These specific DNA fragments as defined by a DNA library then undergo clonal amplification (cluster amplification). This is followed by multiple repeated cycles of labeled nucleic acid sequencing. The resultant DNA reads/fragments are then aligned to the reference genome [2] (Figure 30.1).
In 2000, massively parallel signature sequencing (MPSS) was launched, the first of the NGS technologies which have the capacity to produce billions of small DNA reads per instrument run, with an ability to deliver quick, economical, and reliable genome information. The technology has since been refined many times, resulting in a dramatic reduction of the cost of genomic sequencing [1]. A recent joint recommendation of the Association for Molecular Pathology and the College of American Pathologists, reporting the standards and guidelines for validating next generation sequencing bioinformatics pipelines, has been published [3].
NGS can be useful in rapidly identifying disease-causing mutations and higher-risk alleles, mapping new mutation sites and providing an insight into pathogenic mechanisms, all of which have expanded the diagnostic sphere of rare Mendelian genetic disorders. NGS can identify disease-causing variants, benign genomic variations, and variants of unknown significance. The latter implies insufficient evidence to support pathogenicity of the detected variants [4]. As a result, NGS refined the classification of some neurological disorders through accurate profiling of their molecular basis.
However, NGS poses new challenges pertaining to the analysis, storage, and interpretation of the massive amount of generated data, raising new legal and ethical concerns [4]. For instance, the identification of epigenetic changes, trinucleotide repeats, or changes in non-coding regions will depend upon the number of DNA reads and the coverage. Technically, complete coverage using NGS may never be reached since specific genomic regions such as GC-rich areas and repetitive elements are difficult to amplify and large copy number variations are sometimes not detected.
Whole-Genome Sequencing
Whole-genome sequencing (WGS) allows the high throughput sequencing of the whole human genome. It bypasses the need for targeted capture before sequencing (as seen in whole-exome sequencing [WES]), shortening the turnaround time to obtain results [5]. In the right clinical setting, WGS has reduced the number of tests required for a diagnosis to be established.
The limitation of WGS includes the limited access to WGS technology (especially in a non-specialist setting), relatively high costs, and incomplete coverage of candidate genes. The enormous amount of generated data can be difficult to interpret, and thus WGS has not yet been universally integrated into clinical genetics services.
Whole-Exome Sequencing
WES allows the targeted capture and sequencing of all coding parts of the human genome. It is widely used in the clinic and has become a frontline diagnostic tool for gene discovery and diagnosis of rare diseases [6]. The use of WES has advanced our ability to conclusively arrive at a specific genetic diagnosis in monogenic disorders [7]. For example, in overlapping clinical phenotypes of early-onset generalized dystonia, WES is used to establish or confirm the molecular diagnosis, highlighting the heterogenous genetic causes of this condition [8].
Employing WES as a diagnostic tool in the clinic might be limited by the need for the appropriate system infrastructure, including computer tools, pipelines, and filter for data analysis and storage, as well as expertise in data analysis and appropriate genetic counseling [5]. For example, the finding of variants of unknown significance or pathogenic variance in genes not known to be associated with the phenotype of interest is a significant challenge for clinicians and researchers, to correctly interpret the data. Consequently, the cost-effectiveness and validity of exome sequencing for the diagnosis of complicated neurodegenerative disorders is yet to be established [9].
Custom Targeted Design
The custom targeted design approach involves the sequencing of a variable number of known genes, and is also known as targeted re-sequencing (or gene panels). With targeted re-sequencing, a subset of genes or target regions are tested and sequenced [10]. Commercial panels for a specific group of diseases, e.g. a dystonia panel, or design panels including only specific genes of interest (custom panel) are available at a low cost and with a rapid turnaround time [4, 11]. Gene panels allow an increased coverage and depth of the candidate genes compared to WES and WGS [12]. A major limitation of gene panels is the rapid expansion of gene discovery. Most commercial labs are lagging behind in incorporating new genes into their panels. Therefore, negative testing does not necessarily negate a genetic diagnosis, especially if the gene of interest is not included in the panel.
A comparative summary of WGS, WES, and custom targeted design is provided in Table 30.1.
Table 30.1 A comparative summary of different NGS approaches: WGS, WES, and custom targeted design (gene panels)
| Next-generation sequencing | Summary of method | Advantages | Disadvantages |
|---|---|---|---|
| WGS | High throughput sequencing of the whole human genome |
|
|
| WES | Targeted capture and sequencing of all coding parts of the human genome | Widely used in clinical practice |
|
| Custom targeted design (gene panels) | Subset of genes or target regions are tested and sequenced |
| Due to rapid expansion of gene discovery, newly identified genes are not immediately included in commercially available panels |
Next-Generation Sequencing in Diagnosing Inborn Errors of Metabolism
Inborn errors of metabolism (IEMs) encompass a complex and heterogenous group of genetic diseases with evolving and overlapping clinical phenotypes of varying severity. More than 600 genes for IEMs have been identified to date. Many IEMs are associated with movement disorders (hyperkinetic and/or hypokinetic) [13]. The prompt and accurate identification of the molecular basis of IEMs is crucial for appropriate treatment and genetic counseling.
The implementation of NGS technologies has changed the diagnostic approach and yield in IEMs of childhood [4]. In a study of 146 patients with either a suspected IEM based on clinical and biochemical evidence (n = 81) or a suspected IEM based on clinical evidence and non-specific biochemical markers (n = 65), a genetic diagnosis was achieved in 50% of patients using a custom targeted genes panel followed by Sanger validation. Not surprisingly, the diagnostic yield was higher in the first group (78%) compared to 15.4% in the second group [14].
The diagnostic utility of NGS has proven beneficial in adults with undiagnosed IEMs as well. The diagnosis of IEMs of complex phenotypes or IEMs with episodic presentation and insidious progression is a clinical challenge. Patients with atypical presentations or atypical age of onset are often misdiagnosed for years. NGS can improve the diagnostic yield in patients with atypical presentations and shorten the time to diagnosis. Examples include adolescent-onset Krabbe disease masquerading as multiple sclerosis due to episodic presentation and demyelination on MRI and diagnosed by genetic testing at age 40 years [15] or Pompe disease diagnosed at age 59 years in a woman presenting with a cerebral stroke and left ventricular hypertrophy with a long-standing history of gait disturbance [16].
Use of Next-Generation Sequencing for Genomic Newborn Screening
The current standard of newborn screening (NBS) includes testing for enzyme activity (e.g. biotinidase) or specific molecules on a tandem mass spectrometry platform (e.g. some lysosomal storage diseases, urea cycle defects, organic acid disorders, fatty acid oxidation disorders). In many cases, a positive result by first-tier testing is confirmed by molecular analysis as second-tier testing.
Genomic newborn screening (GNS) is a screening for genetic diseases that can cause death, serious lifelong disability, or chronic disease if not treated shortly after birth. The aim of GNS is to identify conditions for which effective therapy is available and to provide this treatment early enough to prevent or ameliorate complications of a disease. Dried blood spots are considered an appropriate material for future newborn screening programs relying on high-throughput sequencing technologies [17].
The use of genome-wide (whole-genome or -exome) sequencing for population-based newborn screening presents an opportunity to detect and treat or prevent many more serious early-onset health conditions. However, some disorders still have no clear natural history, specific treatment, or age of onset and hence are less ideal candidates for GNS.
NGS in the Diagnosis of Specific Inherited Metabolic Movement Disorders
The diagnostic pathway for metabolic movement disorders of childhood is based primarily on detailed clinical phenotyping; metabolic/biochemical profiling in the urine, plasma, or CSF; and neuroimaging findings. While biochemical profiling has a rapid turnover and is readily available, its diagnostic yield is low. Similarly, neuroimaging findings can be non-specific (e.g. hyperintense signal in the basal ganglia). NGS technologies have been increasingly utilized as first-tier testing in different movement disorders [18], including dystonia [19], cerebral palsy [20], ataxia [21], and Parkinson disease [22].
In this section, we will showcase a few examples of how NGS technology is used to diagnose metabolic movement disorders, to identify novel genetic variants, and to expand phenotypic–genotypic associations.
Dopa-Responsive Dystonia
Dopa-responsive dystonia (DRD) refers to a group of neurometabolic movement disorders of tetrahydrobiopterin (BH4) and monoamine neurotransmitter synthesis that share a favorable clinical response to levodopa treatment. These disorders overlap in the clinical phenotype, but vary in the underlying enzymatic defect and genetic etiology [23].
Traditionally, the diagnostic algorithm of DRD is based on a suggestive history of childhood-onset focal or segmental progressive lower limb dystonia with diurnal fluctuation, a positive family history of DRD, and robust clinical improvement following an adequate 3-month trial of levodopa treatment [24]. In cases with an equivocal response to levodopa, a phenylalanine challenge to determine the phenylalanine to tyrosine ratio can support the diagnosis of DRD but does not identify the underlying enzymatic or genetic defect. CSF testing for intermediate metabolites of dopamine and BH4 synthetic pathways (biopterin, neopterin, 5-hydroxyindoleacetic acid, homovanillic acid) and phenylalanine in the blood can provide signature profiles of abnormalities associated with specific subtypes of DRD [23, 24].
There are five classic DRD syndromes with an identified phenotype–genotype association [23] where the heterogeneity of underlying genetic defects influences the clinical phenotype. These include the following syndromes: (1) Autosomal-dominant GTP cyclohydrolase 1 enzyme deficiency or classic DRD (Segawa disease) is associated with more than 200 pathogenic mutations in the GCH1 gene. This is the most common genetic defect identified in DRD and is associated with a robust response to levodopa treatment. (2) The autosomal-recessive type of GTP cyclohydrolase 1 enzyme deficiency is characterized by neonatal onset of symptoms, hyperphenylalaninemia, and responsiveness to levodopa treatment. (3) Tyrosine hydroxylase deficiency-associated DRD is caused by bi-allelic mutations in the TH gene, with more than 100 mutations described, and it is associated with encephalopathy, autonomic dysfunction, and an incomplete response to L-dopa. (4) Sepiapterin reductase enzyme deficiency-associated DRD is caused by bi-allelic mutations in the SPR gene and is associated with severe encephalopathy and dysautonomia and suboptimal response to levodopa. (5) 6-Pyruvoyltetrahydropterin synthase deficiency is caused by bi-allelic mutations in the PTS gene and is associated with cognitive dysfunction, seizures, and responsiveness to levodopa (Table 30.2).
Table 30.2 Summary of five classic DRD syndromes with identified phenotype–genotype associations (adapted from Wijemanne and Jankovic [23])
| Molecular basis | Gene | Phenotype |
|---|---|---|
| Autosomal-dominant GTP cyclohydrolase 1 enzyme deficiency (Segawa disease) | GCH1 |
|
| Autosomal-recessive GTP cyclohydrolase 1 enzyme deficiency | GCH1 |
|
| Autosomal-recessive tyrosine hydroxylase deficiency | TH |
|
| Autosomal-recessive sepiapterin reductase enzyme deficiency | SPR |
|
| Autosomal-recessive 6-pyruvoyltetrahydropterin synthase deficiency | PTS |
|
Specific gene sequencing for GCH1, TH, SPR, and PTS is considered an appropriate first-tier testing. However, as the repertoire of novel mutations in these genes is expanding, current commercially available panels might fall short of identifying recently described mutations, yielding a false negative interpretation [23, 25].
In cases with an atypical presentation, inadequate response to levodopa, or negative gene sequencing results, WES might be considered to cover mutations not included in the panel testing or to rule out other genetic disorders, including those that may show some response to levodopa such as ataxia–telangiectasia [26], spastic paraplegia type 11 [27], and spinocerebellar ataxia type 3 [28].
With the use of NGS the construct of DRD, therefore, has shifted from a single phenotypic–genotypic association (Segawa disease) to include heterogenous genetic disorders of similar clinical phenotype with significant treatment and management implications. With more than 150 genes implicated in IEMs, including mitochondrial disorders and neurodegenerative conditions identified in patients with childhood dystonia, experts support the strategy of prioritizing NGS in a selected subset of patients to reach a definitive diagnosis in a time-efficient and cost-effective manner [29].
Finally, the implication of a molecular diagnosis using NGS extends beyond diagnosis to therapy. While levodopa is considered the standard treatment in DRD, the heterogeneity of the underlying enzymatic and molecular defects often require additional treatments such as 5-hydroxtryptophan and BH4 for 6-pyruvoyltetrahydropterin synthase deficiency [30].
Related posts:
Chapter 1 – Treatable Metabolic Movement Disorders: The Top 10
Chapter 10 – A Phenomenology-Based Approach to Inborn Errors of Metabolism with Spasticity
Chapter 7 – A Phenomenology-Based Approach to Inborn Errors of Metabolism with Ataxia
Chapter 16 – Syndromes of Neurodegeneration with Brain Iron Accumulation
Chapter 4 – Imaging in Metabolic Movement Disorders
Chapter 28 – Disorders of Creatine Metabolism: Creatine Deficiency Syndromes and Movement Disorders
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
