In the present study, we have described the clinical features, investigations, treatment and outcome of 9 genetically confirmed patients with BH4 deficiency from South India. The disorders of BH4 metabolism are broadly classified into six types based on the specific enzyme defects into autosomal recessive (AR) or autosomal dominant (AD) GTPCH deficiency, PTPS deficiency, DHPR deficiency, SR deficiency, and PCD deficiency. PTPS deficiency is the commonest cause of BH4 deficiency (56.7%), followed by DHPR deficiency (34.7%), GTPCH deficiency (4.9%) and PCD deficiency (3.7%) [5, 14]. SR and PCD deficiencies are mild and rarely described.
Most of the reports on disorders of BH4 metabolism are from western literature, Japan, and China where universal new-born screening for PKU is practised [5-9]. Opladen et al., 2012 has described the largest cohort of 626 patients with BH4 deficiency from the BIODEF database [5]. Unlike previous studies most of the patients in our cohort belonged to DHPR deficiency(66.6%), followed by AR GTPCH deficiency (22.2%) and PTPS deficiency (11.1%). This discrepancy could be because of the referral bias as ours was tertiary/quaternary health care centre. All our patients were diagnosed when symptomatic except one who was detected on new-born screening. Our patients had an median age at onset of symptoms of 6 months and were diagnosed around 15 months (8 months – 120 months) which is delayed as compared to previous studies [6]. Though there is glaring biochemical difference among the various BH4 deficiencies clinical features overlap in most and can mimic cerebral palsy, extrapyramidal disorder or genetic epilepsies. The cardinal clinical features described include hypotonia with developmental delay, cognitive impairment, seizures, autonomic disturbances and movement disorders mainly dystonia, oculogyric crisis, dyskinesias and early onset parkinsonism [3-5]. Other less-described symptoms are swallowing difficulties, hypersalivation, sleep disturbances, psychological issues, prematurity, low birth weight and central hypothyroidism [15]. Hypopigmented skin and hair, characteristic musty body odour, and eczema are few systemic findings [16]. In our cohort neuroregression (89%) was the most common presentation followed by developmental delay (77.7%), dystonia (77.7%), seizures (55.5%) and behavioural problems (22.2%). Extrapyramidal symptoms dominated in patients with GTPCH deficiency, seizures in DHPR and PTPS deficiency, whereas regression and developmental delay was present across the cohort. Ataxia was observed in Patient-1. Consanguinity was high and noted in 89%. Microcephaly (33%), oculogyric crisis (22%), hypotonia (22%) and dystonia (77.7%) were remarkable examination findings. Microcephaly is commonly reported in patients with PTPS and DHPR deficiency, while it is uncommon in GTPCH deficiency[17]. In our study, the patients with PTPS and GTPCH deficiency as well as one patient with DHPR deficiency reportedly had microcephaly. Normally in patients with PKU or BH4 deficiencies there will be decreased skin and hair pigmentation due to reduced levels of tyrosine and competitive inhibition of tyrosine uptake by phenylalanine [18]. But skin and hair changes were not that prominent in our cohort (11%) probably as blood phenylalanine levels were not markedly elevated. Prematurity, low birth-weight, musty odour and autonomic nervous system symptoms was not present in any of the patients in our cohort.
Hyperphenylalaninemia (HPA) is classically defined as plasma phenylalanine levels greater than 120 µmol/l (2mg/dl) [19]. HPA can result either from deficiency of the enzyme phenylalanine hydroxylase or its cofactor BH4. Disorders of BH4 metabolism are rare and contribute to around 2% of the cases detected to have HPA [19]. HPA is seen in almost all the subtypes of BH4 deficiency except in AD-GTPCH and SR deficiency. Only mild HPA was detected in our cohort with highest blood phenylalanine levels pre-treatment being 676 µmol/l. Patient-6 with DHPR deficiency had an increased Phe/tyr of 3.5 with a borderline baseline phenylalanine levels suggesting HPA. Surprisingly, Patient-8 with AR-GTPCH deficiency didn’t have HPA [20]. Ideal investigation after detecting HPA is to assess blood or urinary pterins (biopterin, neopterin, sepiapterin), CSF analysis of neurotransmitters [5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA)], pterins and 5-MTHFR and erythrocyte DHPR activity which will help in diagnostic confirmation and also in differentiating among the various subtypes of BH4 deficiencies [14]. The presence of HPA with normal BH4 cofactor metabolites suggests PKU and excludes BH4 disorders. Unfortunately as ours is a resource limited setting the above mentioned biochemical tests could not be performed in our study.
EEG changes previously described in the literature are non-specific [5,6]. Three patients in our cohort with DHPR deficiency had abnormal EEG findings, Patient-2 had multifocal epileptiform discharges, Patient-3 had classical hypsarrhythmia and Patient-5 had an EEG consistent with ESES. Drug refractory epilepsy was observed in all the three cases with abnormal EEG. Neuroimaging findings in BH4 deficiencies are indeterminate and not routinely recommended for diagnosis [14]. In the cohort by Ye et al., 2012 cerebral MRI abnormalities were seen nearly in 43% and mainly showed cerebral white matter changes[6]. Imaging changes are commonly described in patients with DHPR and PTPS deficiency. Similar to our cohort, the imaging findings described include delayed myelination, signal changes in parieto-occipital white matter, cerebral atrophy and central tegmental tract (CTT) hyperintensities [21, 22] . Intracranial calcifications like in our Patient-6 with DHPR deficiency, mainly in basal ganglia and subcortical regions have been previously described in patients with DHPR deficiency and in only one patient with PTPS deficiency [23]. Intracranial calcifications have been hypothesized to be secondary to cerebral folate depletion in DHPR deficiency. Other imaging findings in BH4 deficiency include subcortical cyst like lesions on T1 weighted images [22]. However, no correlation has been observed with the imaging features and degree of neurological impairment [23]. A total of 11 genetic variants were identified in our cohort with 9 P/LP variants related to BH4 disorders: 6 in the QDPR gene, 2 in the GCH gene and 1 in the PTPS gene. Most of the variants have been previously reported in ClinVar or HGMD except three novel variants of QDPR gene c.296-1G>T, c.295+5G>T and c.680T>C.
Earlier the initiation of treatment in BH4 disorders better is the neurodevelopmental outcome [5,6]. The main target of treatment is to lower the plasma phenylalanine levels by low phenylalanine diet in conjunction with BH4 supplementation, and replacement of monoamine neurotransmitters namely L-Dopamine and 5-HT [14]. Sapropterin hydrochloride, synthetic analogue of BH4 was first approved by FDA in 2007 for the treatment of BH4 responsive HPA [7]. Oral BH4 at therapeutic dose doesn’t cross blood brain barrier and is mainly involved in lowering peripheral blood phenylalanine levels. However oral BH4 is expensive and most of our patients didn’t receive it due to non-affordability and non-availability in India. Only one of our patient was able to access BH4 and received it at a suboptimal dose of 2mg/kg/day later in the disease course. The actual recommended dose of BH4 is around 5-10mg/kg/day. Sometimes a higher dose of BH4 (upto 20 mg/kg/day) is required to maintain optimal CSF BH4 levels [24]. Among patients with DHPR deficiency, traditionally treatment with only low phenylalanine diet and neurotransmitter replacement has been advocated with controversial role of BH4 as the defect is mainly in BH4 recycling. This is because, a very high dose of BH4 (>20mg/kg) will be required if BH4 is solely used for treatment of HPA in DHPR deficiency. This leads to an excess of BH2 which in turn leads to inhibition of aromatic acid hydroxylases, NO uncoupling resulting in oxidative stress and neurotoxicity [25]. Nonetheless Coughlin et al., 2013 has reported clinical improvement in a case of DHPR deficiency with oral supplementation of BH4 (upto 40mg/kg) along with levodopa and 5-HT [26]. Patient-2 with DHPR deficiency who received BH4 at 2mg/kg/day had no adverse effects and the dose was inadequate for any significant clinical improvement.
Levodopa in combination with peripheral decarboxylase inhibitor (carbidopa/benserazide) is recommended at a dose of 3-7 mg/kg/day (GTPCH deficiency) and upto 10 mg/kg/day in rest of the BH4 disorders starting at a low dose of 0.5-1mg/kg/day [14]. All the patients in our cohort received levodopa/carbidopa at a median dose of 3mg/kg/day (range 1-10mg/kg). 5-HT starting at a dose of 1-2 mg/kg/day (target dose 5mg/kg/day) is recommended in all BH4 disorders except AD-GTPCH and PCD deficiency [14]. Only five patients in our cohort received it at a median dose of 3mg/kg/day (range 1-5mg/kg) due to affordability issues. Folinic acid (10-20mg/day) is beneficial in patients with DHPR deficiency as it leads to secondary cerebral folate depletion and also in cases with low CSF 5-MTHFR [14]. Owing to lack of facility for monitoring CSF 5-MTHFR, all the patients in our study received folinic acid at a dose of 10-15mg/day. Additionally children received symptomatic management for seizures and dystonia. Medication related adverse events was not observed in our patients, except P-7 with GTPCH had mild dyskinesias with L-Dopa. Ideally monitoring of treatment by blood/plasma phenylalanine levels, CSF HVA, 5-HIAA and 5-MTHFR and titration of dosage of BH4, levodopa and 5-HT is essential to ensure optimal level of neurotransmitters required for brain development [14]. Prolactin is another indirect marker used for assessing effectiveness of levodopa in order to avoid invasive lumbar puncture [27]. Selective monoamine oxidase inhibitors, dopamine agonists, catechol-o-methyltransferase inhibitors, selective serotonin reuptake inhibitors are the next line agents suggested for treatment of BH4 disorders [14].
We had a median duration of follow up of 15 months in our study, with a median reduction of blood phenylalanine levels by 190 µmol/l. Though biochemical response has been marked, except for patients with GTPCH deficiency, only mild clinical improvement was noted with regards to developmental milestones, seizures or dystonia. Developmental quotient at follow up in 8/9 patients was < 70 suggesting severe developmental delay. This finding is in line with other studies wherein plasma phenylalanine levels had no correlation with the developmental quotient. Rather, developmental quotient inversely correlated with the age of initiation of treatment [6, 23]. Irreversible neuronal damage (basal ganglia calcification, neuronal loss) in DHPR deficiency occurs at a very early age that fails to amend with therapy. Hence, detection of the disorder within first month of life will ensure good neurological prognosis [28]. Patients who were initiated on treatment at early age, had higher incidence of better neurological outcome with lesser children having mental retardation, hypotonia or seizures [5,28]. However, few patients develop severe phenotype despite initiation of treatment at an early age [29]. Children with later age of onset of treatment displayed mental retardation, aggressive behaviour, irritability and autistic features, despite improvement in IQ [23, 30]. Moreover, patients with earlier age of onset of treatment had achieved normal IQ. Although our study comprised of only 9 patients, the mean development quotient of 44.8 ± 28.9 must be interpreted with caution. Yet, it is much lower than the development quotient observed after 3 years of treatment in another study (78±15) [23].
Our data indicates that patients in our follow up have much worse neurological outcome than reported in previous studies [30].There are two factors leading to this wide discrepancy. Firstly, these studies were done in countries where new-born screening for these disorders has been implemented with good coverage, resulting in detection in the neonatal period, leading to early initiation of treatment. Secondly, most of the patients were treated with BH4 supplements unlike our patients, hence affirming the fact that BH4 is indispensable in the treatment for BH4 deficiency. However, these factors may not hold true for all patients. Patient-1 diagnosed and started on treatment at the age of 4 years improved tremendously, while Patient-2 and Patient-5 diagnosed and started on earlier treatment has poor developmental outcome, with drug refractory seizures [28]. The overall outcome of these patients with BH4 deficiency is tremendously conditioned by the age of initiation of treatment and the adequacy of the dose of the medication. Folinic acid administration, particularly plays a very significant role in the developmental outcome [29]. Patients who have developed severe developmental delay and low CSF HVA and HIAA values before starting treatment are unlikely to make complete recovery even with the best medical management [28]. The lack of wide accessibility to pterin and CSF neurotransmitter analysis makes monitoring of treatment highly dependent on clinical assessment, that may not give sufficient evidence for dosage optimization, thereby leading to subtle progressive brain damage [16, 23]. In addition to serum phenylalanine, regular monitoring of serum prolactin may remove impediments to a certain extent in this regard.
Management of BH4 deficiency in India poses a major challenge. The non-availability of diagnostics and lack of national neonatal screening programmes for detection of these disorders is compounded by the lack of BH4 supplements for the diagnosed patients [31]. Universal new-born screening for HPA in conjunction with BH4 cofactor metabolites is essential since 25% of children with PTPS and GTPCH deficiency and 40% of children with DHPR deficiency are asymptomatic in the new born period and remain asymptomatic until 4 months of age [5, 32]. The new born screening may infact be normal on day one of life and hence a repeat testing is essential later after day-3 of life [30]. The first step towards inclusion into the new-born screening programme in India as well as availability of BH4 supplements will pave way for earlier identification and better neurodevelopmental outcome for Indian children with this rare yet treatable neurotransmitter disorder.
To the best of our knowledge, this is the first Indian study described on a cohort of genetically confirmed patients with BH4 deficiency. Strengths of the study includes strict inclusion of only genetically confirmed cases of disorders of biopterin metabolism, elaborate description of clinical features, investigations, treatment, and outcome. Limitations include the retrospective nature with non-uniform data and follow up, lack of facility to perform BH4 cofactor analysis, segregation analysis, parental analysis, and functional studies. Although the study suffers from its inherent limitations, this is a novel endeavour to highlight the challenges faced during management of patients with BH4 deficiency in India. This study also highlights the need for initiation of nationwide new-born screening which is an unmet need of the hour not only for HPA but also for various other inborn errors of metabolism which have a better outcome when detected and treated early.