A diagnosis of Hunter syndrome is usually suspected in young people who display signs and symptoms of the condition. Characteristic changes in the facial features provide the main initial warning that a child may have Hunter syndrome. In most cases, the diagnosis is usually made at around 2 to 4 years of age. Once a diagnosis is suspected, further laboratory tests are arranged by the physician to confirm the diagnosis.

Hunter syndrome is a lysosomal storage disease that is inherited in an X-linked recessive manner. The condition therefore mainly affects male children. The disease is caused by a deficiency in an enzyme called iduronate-2-sulfatase (I2S), which is normally required to break down and replace mucopolysaccharides or glycosaminoglycans (GAGs). In particular, the I2S enzyme is responsible for the breakdown of dermatan sulfate and heparan sulphate. However, deficiency or absence of this enzyme in people with Hunter syndrome means GAGs accumulate in cells throughout the body, particularly in tissues where dermatan sulfate and heparan sulfate are abundant. As increasing amounts of GAGs collect in cells, the function of organs and cells is increasingly compromised, leading to more severe symptoms as the disease progresses.

Further tests that are carried out to confirm the diagnosis include blood, urine or tissue samples. First, a general screening test to check for the presence of a mucopolysaccharide (MPS) disorder is performed using a urine sample. A definitive diagnosis can then be made by checking the I2S enzyme level, which can be determined using blood, white blood cells or a skin biopsy. Analysis of the I2S gene may also be performed to determine the severity of the condition.

A diagnosis can also be made before a baby is born, using prenatal testing. Here, the fluid that surrounds the baby while it is in the womb called the amniotic fluid can be tested for levels of I2S activity using a process called amniocentesis. Alternatively, the activity of the enzyme can be checked using a tissue sample taken from the placenta, which is referred to as chorionic villus sampling.

In some cases, a diagnosis of Hunter syndrome may come to light due to the presence of another health issue. A child may have pneumonia, for example, and a chest X-ray may happen to show ribs or vertebrae that are abnormally shaped, which is a symptom of the condition. However, due to slow progression of the disease and the number of conditions the syndrome shares clinical features with, a definitive diagnosis can take some time to reach.