As a three-part series, we’re exploring how the standard of care, as it pertains to genetic testing, is evolving for rare disease diagnosis. In this first part we’re looking at the traditional approach to genetic testing by following the typical diagnostic journey of a young child with symptoms of developmental delay. Symptoms such as a delay in speech and language skills, social and emotional difficulties, and sensitivity to certain sensations.
Fragile X syndrome testing
One of the first diagnoses considered in a case of a child exhibiting developmental delay is fragile X syndrome. Particularly if the child is male and there is a history of undiagnosed mental retardation in the family. The disorder, caused by mutation of the X-linked FMR1 gene, is the most common cause of inherited mental retardation, with males typically more severely affected than females. In nearly all cases, the causative mutation is an expansion of the unstable CGG repeat sequence present in the 5’ UTR of the gene. FMR alleles are roughly categorized into four classes: normal (up to 40 repeats), intermediate (41 to 60 repeats), premutation (61 to 200 repeats) and full mutation (>200 repeats). Full mutation alleles are typically hypermethylated, resulting in silencing of the gene which leads to no mRNA production and the mental retardation phenotype. The relevance of intermediate alleles is not completely clear, but (due to FMR1’s X-linkage) females carrying one copy of a premutation allele have a 50% chance of passing the allele on to their offspring, with a high likelihood that the number of repeats within the allele will be further expanded during transmission. Males carrying a premutation allele have a 100% chance of passing the allele on to their daughters, but the likelihood of further expansion during transmission is very low.
Fragile X syndrome testing is typically performed by PCR or Southern blot analysis, or a combination of both1. PCR analysis uses primers flanking the repeat region to amplify the spanning DNA. The size of the PCR product is then used to approximate the number of repeats present in each allele. PCR analysis allows for accurate sizing of alleles in the normal, intermediate and premutation ranges, but larger full mutations are more difficult to amplify and may fail to produce a PCR product. In comparison, Southern blot analysis does not allow for accurate sizing of alleles, but does detect alleles of all sizes including full mutation alleles. Unlike PCR analysis, Southern blot analysis can provide additional information about the methylation status. But it may be affected by highly skewed X-chromosome inactivation, which does not affect PCR analysis. Given their relative strengths and weaknesses, a combination of both analyses will often provide the most complete results.
When fragile X syndrome testing fails to identify expansion of the FMR1 gene as the molecular cause behind a case of developmental delay, a typical next step is to analyze the genome for large structural aberrations such as chromosomal gains (duplications) and losses (deletions). Cytogenetic testing via microarray-based comparative genomic hybridization (array CGH) is used for this purpose2.
Array CGH analysis uses a series of probes that hybridize with selected DNA regions. The copy number of each region is estimated by comparing the ratio of probe intensities produced through comparative hybridization of labeled patient DNA to an in silico reference data set. A relatively high probe intensity is indicative of a duplication or gain of chromosomal material, while a relatively low probe intensity is indicative of a deletion or loss of chromosomal material. The design of the microarray, including the number and spacing of probes, will ultimately determine the resolution limits. However, duplications and deletions under 50kb-100kb in size are unlikely to be detected by most platforms.
Targeted gene analysis
When array CGH testing fails to identify large structural aberrations, a typical next step is to look for smaller structural changes, as well as smaller point mutations and indels, that may be responsible for the developmental delay. To detect these types of genetic changes, targeted sequencing or deletion/duplication analysis of one or more candidate genes is used.
Traditionally Sanger sequencing of individual genes would have been used to identify the smaller mutations. More recently, next generation sequencing (NGS) technology has enabled simultaneous sequencing of larger numbers of related genes as part of a targeted panel. However, sequential testing of individual genes or progressively larger gene panels is often still performed. In the case of both targeted gene and panel sequencing, discrete regions of the desired gene(s) are amplified, sequenced and analyzed for single nucleotide variant (SNV) changes and small (<20bp) indels. High density targeted arrays, as well as some types of panels, may be used separately or in conjunction to identify larger structural variants (>20bp) affecting the specific gene(s) being tested.
In the second part of the series we’ll be looking at how the standard of care is evolving, with whole exome sequence (WES) testing increasingly being accepted as an alternative to targeted gene analysis, for faster and more cost-effective identification of causative SNVs and indels.
1. South ST, et al. ACMG Standards and Guidelines for constitutional cytogenomic microarray analysis, including postnatal and prenatal applications: revision 2013. Genet Med. 2013 Nov;15(11):901-9. PubMed ID 24071793
2. Sherman S, et al. Fragile X syndrome: diagnostic and carrier testing. Genet Med. 2005 Oct;7(8):584-7. PubMed ID 16247297