Practical guide to genetic testing in pediatrics

If you are not a Geneticist and you have to approach the world of genetic testing, you may have some difficulty in choosing the best test for your patient.

In recent years, thanks to the advancement of Next-Generation Sequencing (NGS) technologies, the lowering of their costs and the increase in knowledge about genetic diseases, the execution of a genetic test is increasingly becoming one of the cornerstones of the diagnostic process.

But what is the most appropriate test for my patient’s phenotype? Which is the fastest and cost-effective?

Remember: the right choice will allow you to solve your patient’s diagnostic odyssey in the shortest time!

Let’s take a step back…

The Italian guidelines require that any genetic test is prescribed only by a Geneticist, following thorough genetic counseling, in which are investigated the patient’s family history, his medical history, the presence of physical signs and all other things which may be useful in clarifying the diagnostic suspect.

In other parts of the world, it is possible that the prescription of the genetic test is also performed by other healthcare professionals, who may not have the sufficient skills to choose the correct test for a certain patient.

Here is some information on some of the main genetic tests used, their applications, limitations, turnaround time (TAT) and performance.

In general, the choice of the test is closely linked to the patient’s clinical presentation. Is the phenotype highly suggestive for a monogenic disease? In this case, thanks to low costs and times, it is worthwhile to test the single gene. Does the patient have a disease that is difficult to diagnose? Better to proceed with whole-exome sequencing. Could the patient have a chromosomal rearrangement? Let’s not forget the karyotype…

Cytogenetic tests

1. CMA (Chromosomal MicroArray)

CMA is a non-specific technique that allows the identification of large deletions/duplications throughout the genome, and which is used as a gold standard for the analysis of pathologies such as neurodevelopmental disorders, autism or mental retardation and multiple congenital malformations. The CMA has a resolution of about 25Kb (depending on the average distance of the probes), a diagnostic yield ranging from 15% to 20% and a TAT of 2-6 weeks. The CMA has in many ways replaced the karyotype, thanks to the higher resolution and diagnostic yield, but it is important to remember that it is not able to identify balanced chromosomal rearrangements and CNVs of size <10Kb.

2. Karyotype

The karyotype (or chromosomal mapping) allows the visualization of the map of the chromosomes of a cell, and is used to identify chromosomal aneuploidies (such as trisomy 21) and the presence of large chromosomal rearrangements such as translocations and inversions (also balanced). It is also used to assess reproductive risk in families with a positive history for aneuploidy or translocations or in the case of recurrent miscarriages. It has a fairly low diagnostic yield (~ 3%) and also a low resolution (3-5 Mb). The TAT is 1-2 weeks.

Sequencing test

1. Sequencing of a single gene, a panel of genes, or whole exome

If initially it was much less expensive and more efficient to sequence a single gene or a few genes depending on the patient’s phenotype, today genetic diagnostics is increasingly moving towards the application of whole exome sequencing (WES).

Single gene sequencing can be applied in case there is a strong diagnostic suspicion for a monogenic disease (i.e. Cystic fibrosis), with fairly short response times (1-4 weeks) and a diagnostic yield that can reach 100 % (depending on the pathology). The main problem is that very few conditions are monogenic.

The next step in terms of size is the sequencing of a group of genes (panel) related to a specific phenotype. The panels can be pre-established or customizable, have short TAT (2-6 weeks) and are recommended when the clinical suspicion is strong and the differential diagnosis short. The biggest problem with target panels is that they are “immutable” and in case a new gene is discovered for that phenotype they have to be redesigned. For this reason, in many laboratories, including Breda Genetics, the application of “virtual panels” is preferred, starting from data that derive from whole exome or whole genome sequencing.

Whole exome sequencing allows the identification of small sequence variations in the coding regions of the genes. Moreover, recently, many laboratories have developed specific algorithms to be able to identify the CNVs starting from the sequencing data, allowing to further increase the diagnostic yield of the WES (which is around 30-45%). WES is recommended in cases where the clinical suspicion is undefined or there is a long list for differential diagnosis. The TAT is longer than that of the previous analyzes (5-12 weeks) and the cost is also higher. The main limitations are the increased probability of identification of VUS (variants of uncertain significance) and the difficulty in interpreting non-coding variants, as well as the possibility of encountering incidental findings.

2. Sequenziamento dell’intero genoma (WGS, whole genome sequencing)

Whole genome sequencing can be used as an alternative to WES or when WES is negative. In fact, it allows identifying not only the exonic variants but also the intronic ones, thus increasing the diagnostic yield. In addition, it also allows you to identify structural rearrangements and accurately map breakpoints. The main limitations of the WGS, besides the cost which is quite high and the TAT which increases significantly (4-16 weeks), are the difficulty in handling a large amount of data and in interpreting numerous variants (VUS).

Other tests

1. Triplet expansion

Some diseases are due to the expansion of the number of a triplet found in a particular region of the gene. In these disorders, the number of triplets can be quite variable and is divided into 3 ranges: a normal range, a premutation state and a mutation range, in which the disease is overt. Unfortunately, NGS sequencing cannot provide reliable data in these highly repeated regions and specific tests are needed to assess the number of triplets present. The main limitation is that it is necessary to know a priori the patient’s diagnosis in order to proceed with the molecular confirmation. The TAT is 2-4 weeks.

2. Methylation pattern analysis

Other disorders are characterized by an abnormal pattern of methylation of some control regions of the gene. Since these are epigenetic modifications, they cannot be identified by WES alone and specific techniques such as MS-MLPA are instead required. This technique is recommended in the case of suspected imprinting disease or uniparental disomy, has a TAT of 2 to 4 weeks and a variable diagnostic yield.


Taylor A, Alloub Z, Tayoun AA. A Simple Practical Guide to Genomic Diagnostics in a Pediatric Setting. Genes (Basel). 2021 May 27;12(6):818. doi: 10.3390/genes12060818. PMID: 34071827

Yang M, Xu B, Wang J, Zhang Z, Xie H, Wang H, Hu T, Liu S. Genetic diagnoses in pediatric patients with epilepsy and comorbid intellectual disability. Epilepsy Res. 2021 Feb;170:106552. doi: 10.1016/j.eplepsyres.2021.106552. Epub 2021 Jan 7. PMID: 33486335.


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