Neonatal screening for phenylketonuria

Every newborn should be screened by neonatal screening (5) for phenylketonuria. Neonatal PKU screening is a method that allows early detection of hyperphenylalaninemia in an apparently healthy newborn.

Diagnosis by neonatal screening allows early specific treatment to be instituted from the first days of life and consequently the prevention of neuro-psychomotor disability induced by this condition (6,7,8,14,19).

In Romania, neonatal screening for phenylketonuria is carried out at national level in 5 regional centres, an activity funded by the Ministry of Health within the framework of the National Women and Child Health Programme (15, 20, 21).

To perform neonatal PKU screening, the doctor must obtain the mother's informed consent, which is written on the observation sheet.

In order to give consent, the doctor must inform the mother about the value of the screening test, the screening procedure, the significance of a positive result, the steps to take in the event of a positive test. Informed consent may be specifically for neonatal PKU screening or it may be included in the general consent for all maternity investigations including screening tests.

The collection of the PKU screening sample must be recorded by the doctor in the newborn's observation sheet with the date of collection. Mentioning in the newborn's discharge note that screening has been carried out is information to be taken into account in the event of subsequent medical events that call into question the differential diagnosis with congenital hypothyroidism.

The timing of the newborn PKU screening sample collection is dependent on the newborn's gestational age and health status. 

• In normal-weight newborns, the screening sample should be taken 48-72h after birth, (1) or even 24h if phenylalanine measurement is accompanied by phenylalanine/tyrosine ratio determination (2). 
• In underweight or newborn infants initially fed glucose and electrolyte solutions without protein (amino acids), your doctor recommends sampling 3-5 days after the introduction of milk feeding.
• In premature babies, two samples are taken, one a few days after birth and the other at discharge from the maternity ward.

If the newborn requires transfusion, the blood sample for screening should be taken within the first 24 hours of life and repeated after 10-14 days (2).

The increase in plasma phenylalanine, a necessary element in the diagnosis, is dependent on the introduction of milk feeding. In newborns fed without protein, there is no phenylalanine intake necessary for the increase in plasma phenylalanine. Premature infants have an immaturity of the enzyme system which can lead to a transient increase in phenylalaninemia and thus a positive screening test. As the enzyme system matures, plasma phenylalaninemia values normalize.

The dry spot screening method is considered the most effective method of neonatal screening in PKU (5). The screening sample is collected on the Guthrie strip from the baby's heel.

The rapid dispatch of the samples to the laboratory allows the diagnostic process to be accelerated and thus the treatment to be instituted.

If phenylalanine values on the screening test are >2mg/dl or over 120 µmol/L the test is considered positive and the coordinating doctor of the screening centre should inform the family and refer the child to a PKU diagnostic and treatment centre for confirmation of the diagnosis (4). Increased phenylalanine on the screening test raises suspicion of the diagnosis of phenylketonuria.

Positive screening test requires confirmation of the diagnosis in a specialist laboratory.

 

Diagnosis of phenylketonuria

A positive neonatal screening test for PKU must be confirmed diagnostically by plasma phenylalanine assay. The screening test is only an indicative test, as there is a possibility of false positive results.

The clinician may also choose to combine phenylalanine, tyrosine and phenylalanine/tyrosine ratio determinations. Tyrosine and phenylalanine/tyrosine ratio assays together with plasma phenylalanine are investigations that add to the diagnosis, especially in newborns with feeding problems.

A child who has plasma phenylalanine above 360 m/mol/l (increased) is diagnosed with hyperphenylalaninemia and should be treated immediately.

If a child with phenylketonuria is untreated or undertreated, severe neuropsychological clinical manifestations, microcephaly, hypo- and hyper-excitability, seizures occur. With age, intellectual and behavioural development is severely impaired, with children showing anxiety, aggression, autism, low IQ, difficulties in information processing, memory impairment.

Establishment of phenylalanine tolerance is done under inpatient conditions under careful dietary supervision.

1. According to plasma phenylalanine the doctor classifies the patient into the following clinical forms:

• Classic PKU if plasma Phe is > 1200 micromoles/L;
• PKU variants if plasma Phe is between 600 and 1200 micromoles/L;
• mild hyperphenylalaninemia with Phe below 600 (360) or classic PKU with plasma phenylalanine > 1200;
• moderate PKU between 900 and 1200 micromoles/L;
• mild PKU between 600-900 micromoles/L;
• mild hyperphenylalaninemia < 600 micromoles/L.

2. According to the level of phenylalanine tolerance the clinical forms are:

• Classic PKU with a phe tolerance of <21 mg/kg/day;
• moderate PKU with a phe tolerance of 21-50 mg/kg/day;
• Mild PKU with a phe tolerance of > 50 mg/kg/day

3. According to the response to Sapropterin the clinical forms are:

• PKU;
• BH4 deficiency with level below 360 micromoles/L that does not need to be treated and above 360 micromoles/L that needs to be treated.

In turn the PKU patient is divided into PKU responsive or non-responsive.

The doctor may recommend molecular genetic investigations to identify DNA mutations, which is particularly useful if the need to start treatment is unclear. Although molecular genetic investigations are considered adjuvant in diagnosis, they are of epidemiological importance and in determining therapeutic attitude.

Children with hyperphenylalaninemia should be tested for BH4 deficiency.

Detection of BH4 cofactor biosynthesis and recycling deficiencies is essential in determining therapeutic management as children with this condition do not respond to dietary treatment. In this situation neurotransmitters (dopamine, serotonin precursors) and synthetic BH4 (Sapropterin) should be administered. This treatment positively influences the patient's condition. The doctor may opt for BH4 screening by blood/urine pterin and dihydropterin reductase (DHPR) activity on blood spot or by Sapropterin loading test.

In patients with BH 4 deficiency, pterin changes are identical in blood and urine. Blood spot analysis (DBS) is more practical and allows measurement of pterins, DHPR activity and amino acids in a single sample.

The physician may recommend dry spot PKU testing of children with mental retardation, behavioural disorders and deficient pigmentation who have not previously been included in the neonatal screening programme for PKU, as well as siblings and parents of children diagnosed with PKU (especially if the physician detects the presence of psycho-intellectual deficiencies).

Even children diagnosed late can benefit from dietary and drug treatment, where behavioural disorders such as aggression can be improved.

Women with phenylketonuria should receive information and dietary advice before pregnancy. Children of PKU mothers who do not have metabolic control (serum Phe level 120-360 micomol/l) throughout pregnancy may present at birth: congenital heart defects, microcephaly, dysmorphism, intrauterine growth retardation. A maternal Phe level above 1,200 micromol/l greatly increases the risk of congenital anomalies in the baby.

Sapropterin sensitivity testing is recommended for children with hyperphenylalaninaemia and PKU who have reached the age of 4 years. The results of the test may influence the therapeutic attitude towards introducing Sapropterin along with diet in the treatment resulting in increased phenylalanine tolerance, reduced dietary restriction and improved quality of life.

Genotyping can be performed to highlight the response to Sapropterin. Genotype may be predictive of Sapropterin response, but the genotype-phenotype correlations thus identified are imperfect.Sapropterin response is not accurately predicted by genotype except for those with two null mutations in trans. The clinician will recommend testing PKU patients with a daily dose of Sapropterin of 20 mg/kg body weight under the condition of a constant protein intake throughout the duration of testing. A greater decrease in plasma Phe levels was observed at doses of 20 mg/kg/day compared to 5 -10mg/kg/day.

The clinician should consider a patient responsive to Sapropterin if after loading with BH 4 the plasma phenylalanine concentration is found to decrease by at least 30% or phenylalanine tolerance doubles. In patients with concentrations less than 400 micromoles/L responsiveness should be tested only after reaching plasma Phe levels > 400micromoles/L by increasing dietary phenylalanine intake.

The clinician may increase phenylalanine intake by liberalising the diet during the test period provided that protein intake is kept constant (maintaining the same daily menu) or by adding one egg daily or by adding phenylalanine.