3.7 DNA testing

The first DNA test that is done when a child is thought to have an intellectual disability checks for the absence or duplication of chromosomal material. Chromosomal imbalance will show up on a test called 'chromosomal microarray analysis'. “Microarray” refers to a microchip-based testing platform that allows automated analysis of many pieces of DNA at once. Computer analysis is used to compare a patient's genetic material to that of a reference sample. .⁴⁴ 

Because the DNA-changes that result in Pitt Hopkins syndrome are often caused by chromosomal imbalances, we think that this is the right test to start with for people suspected of having PTHS. If it looks like someone has either Pitt Hopkins or Angelman syndrome, we recommend doing another test as well, called 'methylation analysis', specifically of the part of the DNA where methylation-mistakes can cause Angelman syndrome. 

DNA Methylation
'DNA Methylation' is a process by which gene transcription is suppressed. When a part of the DNA is methylated, this means that it can't be copied to make proteins from. It is important that the right parts of the DNA are methylated at the right points in time, otherwise problems can occur, such as in Angelman syndrome.

In some hospitals and laboratories, Next generation sequencing (NGS) can be done. Using NGS an entire human genome can be sequenced within a single day. Genome is the word for all the DNA of one individual, the whole cookbook so to speak. If NGS testing is available, this should be the next step in DNA testing for Pitt Hopkins syndrome, as there are several syndromes that resemble Pitt Hopkins syndrome that can be checked for with this single test.

If Next generation sequence is not available, if the clinical suspicion of PTHS is very strong, a number of tests can be done that only check the part of the DNA coding for Transcription Factor 4. (Figure 3). Sometimes, these tests don't show up changes in TCF-4, because they look at the wrong chromosome. This can happen in case a part of a chromosome broke off and attached itself to another chromosome. To check for this, the chromosomes should be made visible with a technique called 'classic karyotyping'.

FIGURE 3 Molecular diagnostic pathways for Pitt-Hopkins syndrome (PTHS). Two pathways are depicted, one for an individual clinically suspected to have PTHS and one for individuals without this suspicion. As high throughput analyses are not available worldwide, evaluation using Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) is also depicted. The clinical diagnostic criteria are those provided in Table 2

When mutations in TCF4 are found, these should be reported along with a person's clinical characteristics according to the criteria described by the American College of Medical Genetics (ACMG)  (R5). 

If individuals look, act, and develop in ways typical of Pitt Hopkins syndrome, and changes are found in their DNA that probably lead to problems in the functioning of Transcription Factor 4, it is safe to assume these DNA changes caused Pitt Hopkins syndrome. In these cases, parents don't need to be tested to diagnose their child, but we recommend testing them anyway to make sure that the change was 'de novo', meaning that they have almost no chance of having another child with Pitt Hopkins. 

When changes in the DNA are found in areas other than the ones that typically lead to malfunctioning TCF-4, the diagnosis is not as straight-forward, because we know some individuals have such changes without actually looking, acting, and developing as would be typical in case of Pitt Hopkins. The clinical criteria should be checked carefully and it is also useful to check information about variants that have been previously been reported. It is possible to do more testing to check for the effect of the variants on the production of the protein TCF-4. 

One other thing to consider is mosaicism. Mosaicism means the presence of two different sets of DNA (two different cookbooks) in one individual. Normally, all our cells contain the same cookbook, the same DNA. But sometimes, part of our cells contain a variation of the information in the rest of the cells. This situation has also been reported for some people with Pitt Hopkins syndrome. ²⁴. It is especially something to consider in people whose skin is a different color in some places (this is called a pigmentation anomaly).