Antioxidant therapy in a patient with Hyperprolinemia type I presented with mild neuromotor retardation and speech disturbance


 Background: Hyperprolinemia type 1 (HPI) is an autosomal recessive inborn disease caused by mutations/deletions of PRODH gene, which is located on chromosome 22q11. The clinic spectrum involves mainly delayed psychomotor development, mild-to-severe mental retardation, neuropsychiatric symptoms and epilepsy. Although HPI can easily be diagnosed in patients undergoing metabolic screening tests, there is no effective therapy protocol in use. There are studies showing that it does most of its clinical findings by disrupting mitochondria function. Case report: We present a long-term follow-up of a four-year-old girl with mild neuromotor retardation and speech disturbance, diagnosed with HPI and treated with antioxidant therapy (vitamin C, CoenzymeQ10, vitamin B complex and L-carnitine) for six years. It has been shown that antioxidant therapy decreases proline levels properly and provides clinical improvement.


Introduction
Hyperprolinemia type I (HPI) (#239500) is one of the two inherited metabolic disorders resulting from defects in the proline catabolic pathway [1]. The rst step in proline catabolism is catalyzed by proline dehydrogenase (PRODH), a avoenzyme localized at the inner mitochondrial membranes that converts proline to proline 5-carboxylate (P5C). In the second step, delta-1-proline-5-carboxylate dehydrogenase (P5CD) catalyzes the conversion of P5C to glutamate (Fig. 1). The de ciency of the P5C and P5CD enzymes leads to hyperprolinemia type I and type II, respectively. PRODH is located in the 22q11 chromosomal region, where microdeletions, which are responsible for DiGeorge (velocardiofacial) syndrome, can present with psychiatric disorders [2]. Clinical ndings that develop due to HPI vary.
Although genotype/phenotype evaluations have been determined for certain mutations, there is still little evidence [3]. Symptoms of HPI include delayed psychomotor development, mild-to-severe mental retardation, epilepsy, neuropsychiatric symptoms, neurosensory hearing loss, and renal abnormalities. Progressive speech deterioration, sleep disturbance, autistic features, stereotypic behaviors, hyperactivity and schizophrenia have also been reported [1,4,5]. In addition to the effect of high proline levels, the PRODH gene has direct and indirect regulatory effects on mitochondrial functions [6][7][8][9].
There is no de nite treatment protocol yet, and symptomatic treatments are recommended.
Proline-restricted diet and supplementation with vitamin B6 are not effective. Here, we report the longterm clinical and laboratory outcomes of a girl diagnosed with HPI who was treated with antioxidant therapy.

Case
A four-year-old girl was admitted to the hospital with complaints of speech and neuromotor developmental delay, attention de cit and learning disability. She was born to a consanguineous parent by caesarean section without any perinatal complications. Her birth weight was 2900 grams.
Developmental history revealed that her neurodevelopmental milestones were delayed; she began to walk at 23 months of age and spoke her rst words at the age of 32 months. In clinical examination, her ne motor development was below the normal level for her age, and her speech development was poor (i.e., she only spoke 2-word sentences, and she was talking with incomprehensible words). However, she could understand basic instructions. There was no evident autistic symptom in her behavioral examination or on parental reports. The score of the IQ test (Stanford-Binet Intelligence Scale) requested for the evaluation of her mental capacity was 68, which pointed to mild mental retardation. There was no additional systemic nding. Routine biochemical tests were normal, but amino acid analysis revealed high serum proline 892 µmol/L and 789 µmol/L (n: 59-369), respectively. While the lactate level was within the normal range, the alanine level was slightly elevated by 638 µmol/L (n: 152-547). Brain magnetic resonance imaging (MRI) was normal. PRODH gene sequence analysis was performed by using the MiSeq next generation sequencing (NGS) platform, an FDA-approved diagnostic system (Illumina, San Diego, CA, USA). We found that NM_016335.4:c.1357C>T (p. Arg453Cys) (rs3970559) homozygous mutation in the PRODH gene. In the evaluation of the family members, it was found that the mother of the case was being followed up with the diagnosis of multiple sclerosis, and she had demyelinated plaques in her brain MRI. Further examinations of the mother showed that she had borderline intelligence with 78 IQ, and her blood proline level was as high as 793 µmol/l. Genetic screening of the family revealed that the mother was homozygous and the father was heterozygous for the same mutation. In her sister and father, the plasma proline levels were in the normal range, and there were no clinical symptoms.
We initially supported a low-protein diet, but no biochemical or clinical improvement was observed. After obtaining informed consent from the patient and her family, we started antioxidant therapy with 100 mg/day coenzyme Q10, B complex (B1+B2+B6+B12), 500 mg/day vitamin C, and 500 mg/day Lcarnitine. The proline values in the third and sixth months of treatment were 273 and 400 µmol/l, respectively, almost falling to normal limits. Alanine also reached the normal limit and never rose again.
In the follow-up period, proline levels were observed to increase twice in short periods of discontinuation of treatment (Fig. 2). To observe the probable effect of this treatment approach on the serum proline level, we ceased antioxidant therapy for one month and observed that the proline levels gradually increased to the levels of diagnosis range. It was found that speech impairment increased and ne motor skills (eg writing) was impaired dramatically during periods of cessation of treatment. The proline levels of our patient, with and without antioxidant treatment were evaluated and a signi cant difference was observed (p<0.005) (Table). At 69 months of treatment, when she was 9 years and 9 months of age, IQ assessment with the Wechsler Intelligence Scale for Children (WISC-R) showed a verbal score of 70, a performance score of 95, and a full-scale IQ score of 81. Her speech disturbance was improved. No side effects of the drugs were observed during the treatment.

Discussion
We presented a long-term follow-up of a patient with a homozygous PRODH gene mutation and mild clinical manifestations who showed an important decline in plasma proline levels and a slight improvement in clinical symptoms with an increase in intelligence scores after antioxidant therapy.
The type of the mutation or the length of deletion can determine the clinical variability and severity of the disease and the levels of serum proline. High serum proline levels are related to serious clinical ndings, such as epilepsy, severe neuromotor retardation, and behavioral problems [10]. The presence of mild and atypical clinical symptoms, such as speech disturbance, mild neuromotor development delay, attention de cit, and learning disability, can be related to mildly elevated proline levels. The proline levels of our patient are moderately high and may be associated with mild clinical ndings and even good therapy response. . There are con icting reports in the ClinVar database from benign variant to pathogenic [3]. As our case does not have a classical clinical phenotype, unlike previous reports, this variant is most likely patological variant but does not cause severe clinical symptoms. The mother of our patient also has mild developmental delay, learning di culties, and cranial MRI with white matter involvement and the same genotype as her daughter. We see that the older sister and the father, who have no clinical ndings and whose proline levels are within normal limits, are heterozygous.
Although the exact pathophysiologic mechanism is not yet known, several mechanisms have been suggested to explain how high proline concentrations can affect brain functions. In the literature, the most commonly emphasized and proposed mechanism is the alteration in glutamatergic homeostasis. Glutamate is an excitatory neurotransmitter that is important in normal brain function. Glutamate excitotoxicity has been shown to be linked with mitochondrial dysfunction, with energy impairment and subsequent partial membrane depolarization with resultant relief of magnesium blockage of the Nmethyl-D-aspartate channel, which results in excessive in ux of Ca+2. It has been suggested that hyperprolinemia by causing a reduction in glutamate reuptake and Na+, K+ ATPase activity leads to glutamatergic excitotoxity, which leads to pathological signaling and in turn contributes to cell injury and death via the production of free radicals [11]. This cell death may lead to cellular damage in the brain.
It has been shown that increased proline levels induce oxidative stress in rat brains [12] as well as alter glutamatergic transmission at hippocampal synapses [13]. Crabtree et al. found that L-proline is a GABA (gamma aminobutyric acid)-mimetic and can act at multiple GABAergic targets, but disease-relevant concentrations lead to disturbances in GABAergic production by blocking GAD blockage. Researchers suggested that this disturbance in the GABAergic system leads to the accumulation of neuroactive metabolites that cause molecular and synaptic dysfunction and nally psychotic disorders. Abnormalities in the GABAergic system have been shown in many neurodevelopmental disorders, such as attention-de cient hyperactivity disorder and schizophrenia [14].
Recent studies mentioned that low PRODH activity not only causes hyperprolinemia but also has additional effects on the electron transport chain (ETC). PRODH directly and indirectly regulates the ETC [10]. Hancock et al found that PRODH binds directly to coenzyme Q1 and that CoQ1-dependent PRODH activity requires functional Complex III and Complex IV, so PRODH supports respiration independent of Complex I and II activity. It has been shown that in the presence of increasing CoQ1, PRODH activity increases in living cell cultures [15]. We have no additional laboratory ndings of mitochondrial dysfunction in our case. A negative correlation with proline levels and antioxidant therapy was demonstrated. In addition to laboratory improvements, it can be accepted that there is objective progress in speech disturbance and intellectual disability.
We may speculate that antioxidant therapy (including coenzyme Q, carnitine, B complex) has an important in uence on the activity of PRODH, and this treatment approach decreases proline levels and provides optimal acting ETC. Further studies with larger sample sizes are needed to evaluate the clinical and laboratory e ciency of antioxidant therapy in patients with HPI.   Figure 2 Proline levels of patient with and without antioxidant therapy