Laboratory and instrumental examinations

Neuroimaging findings

There are three cardinal neuroradiological features of AGS: cerebral calcification, white matter abnormalities and cerebral atrophy. 
Cerebral calcification (deposits of calcium) is typically located bilaterally in the basal ganglia (particularly the globus pallidus, putamen, caudate nucleus), frequently in the cerebellar dentate nuclei, and often in the frontal and parietal white matter too; however, it can also be present in less common locations such as the periventricular white matter or cerebral cortex. The deposits may be punctuate or larger and more confluent. Calcification can be absent at the onset of the symptoms and instead develop over time, but it is almost always evident at the time of the diagnosis; subsequently it tends to remain stable, even though progression of calcification has been observed in a few cases.   
Even though brain computed tomography (CT) scanning is the most specific neuroradiological diagnostic procedure, cerebral calcification can be identified using various neuroimaging techniques:
-    Brain ultrasound scan. This examination correlates well with the brain CT scan and is particularly useful in newborns/infants, during the period when the anterior fontanelle is still open. However, it is limited by its low specificity, as an ultrasound scan cannot accurately distinguish calcification from other very hyperechoic areas due to bleeding, swelling, infection or inflammation, for example. 
-    Brain magnetic resonance imaging  (MRI) using T2*-weighted and gradient echo sequences that allow greater sensitivity in detecting calcification, yet without reaching the levels obtained with brain CT scans, given that positive gradient echo signals can also derive from iron deposits or bleeding. To overcome this problem a new 3D gradient echo technique has been introduced (susceptibility-weighted imaging, SWI) that, having a high spatial resolution, allows calcification to be detected with the same precision as with CT. 
It is important to be aware that in the presence of a clinical picture suggestive of AGS, the presence of cerebral calcification constitutes a very important diagnostic sign. It should carefully be sought, using appropriate neuroimaging techniques, in all cases of unexplained leukoencephalopathy. It is also important to remember that, at least at the onset of the disease, the presence of calcification should not be regarded as an indispensable diagnostic criterion, given that it may not appear until a later stage of the disease.
 Another typical feature of the syndrome is the presence, in more than 90% of cases, of white matter abnormalities (leukodystrophy). Generally these changes are widespread, confluent and symmetrical and they sometimes show a tendency to cystic degeneration. They are located mainly in the periventricular areas and in the deep and subcortical white matter, sometimes showing a widespread and sometimes an anteroposterior distribution. 
These abnormalities are detectable as hypodense areas on brain CT scans and as hyperintense areas on T2-weighted brain MRI.
Cerebral atrophy is the third cardinal neuroradiological feature of AGS, present in the vast majority of patients (around 92% of the cases reported in the literature). It remains stable or tends to progress, as shown by cases followed up over time. It is located mainly at the level of the frontal and/or temporal lobes where it shows a distinctive simple spiral pattern; the brainstem, cerebellum, basal ganglia and corpus callosum may often be atrophic, too. The cerebral atrophy is a consequence of the white matter alterations and loss, and does not usually involve the cerebral cortex.
In addition to the classic triad of neuroradiological findings, there are also reports of cases with cerebrovascular disease findings: there exist reports, exclusively in patients with mutations in the SAMHD1/AGS5 gene, of episodes of stroke due to stenosis of the cerebral arteries, in some cases reminiscent of Moyamoya syndrome (occlusive vascular disease with the formation of compensatory collateral circulations), and/or to cerebral aneurysms. Other vascular events have been reported in rare cases with mutations in the TREX1/AGS1 gene: thrombosis of the perforating vessels in a child with antiphospholipid antibody syndrome, and cerebral haemorrhage in a child without the above-mentioned vascular abnormalities.
Bilateral striatal necrosis is the typical neuroradiological pattern in individuals with mutations in ADAR1/AGS6. It consists of a bilateral, symmetrical and rather selective lesion involving structures also referred to as basal ganglia (caudate nucleus, putamen and globus pallidus, although the latter is less frequently involved). It can arise at different times, even after the first year of life, and it is characterised in the acute phase by swelling of the structures involved and in the later stages by a loss of volume; both these features are clearly evident on CT scans, but much better visualised on brain MRI. 

Cerebrospinal fluid

 Analysis of the CSF is a useful part of the diagnostic work-up in AGS as it can reveal specific abnormalities: in particular, CSF lymphocytosis (increase in white blood cells in the CSF, with values, varying from subject to subject, ranging from 5 to 100 cells/mm3) and increased levels of IFN (>2 UI/ml); increased levels of IFN can also be found in the blood plasma, albeit inconsistently and with absolute values that are lower than those measured in the CSF (the diagnostic value of this finding is therefore less specific). Both the lymphocytosis and the increased IFN concentrations in the CSF are very  marked at the onset of the disease and in the course of the first year of life, after which, over time, they tend to regress gradually to reach normal levels between the ages of 3 and 4 years. 
Furthermore, 2003 brought the first reports of alterations of certain CSF metabolites, particularly pterins (found to be increased) and folates (reduced). The increase in CSF neopterins has since been confirmed in many patients with AGS and is now considered another marker of the disease, in which it is probably a consequence of the primary overproduction of IFN.

Interferon signature

It has recently been shown that the transcription level of certain genes stimulated by IFN (interferon-stimulated genes) is increased in the peripheral blood of patients affected by AGS,  and remains increased for a prolonged period of time, longer than the duration of the increased concentration of IFNin the CSF. 
This increased expression of the interferon-stimulated genes is referred to as the “interferon signature” and it is a very valuable biological marker of the disease, for two reasons: first, it is easily measurable with minimally invasive methods, such as a simple blood test, and second, as mentioned, it remains high for many years, after the CSF level of IFNhas already returned to normal and the phase of clinically evident disease activity has passed. It is important to underline that the interferon signature is positive in the vast majority of patients with mutations in the AGS1 and AGS3-7 genes, whereas it is negative in at least 25% of individuals with mutations in AGS2/RNASEH2B; for this reason, absence of the interferon signature does not exclude a diagnosis of AGS.