Chapter 22 – Neurotransmitter Disorders: DNAJC12-Deficient Hyperphenylalaninemia – An Emerging Neurotransmitter Disorder

Abstract

DNAJC12 deficiency is a recently described form of hyperphenylalaninemia (HPA) [1]. HPA represents a metabolic condition caused by a deficiency in either phenylalanine (Phe) hydroxylase (PAH) or in one of the enzymes involved in the biosynthesis or regeneration of tetrahydrobiopterin (BH4) [2]. BH4 is the natural cofactor of PAH, tyrosine hydroxylase (TH), tryptophan hydroxylases (TPHs), and alkylglycerol monooxygenase, as well as all isoforms of nitric oxide synthase [3].

Chapter 22 Neurotransmitter Disorders: DNAJC12-Deficient Hyperphenylalaninemia – An Emerging Neurotransmitter Disorder

Nenad Blau and Manuel Schiff

Introduction

Variants in DNAJC12 lead to mild HPA, biogenic amines deficiency in the central nervous system, causing intellectual disability, dystonia, and parkinsonism, thereby defining a new entity of HPA without PAH or BH4 deficiency [4]. This expands the clinical and metabolic spectrum of patients detected in the newborn screening for phenylketonuria (PKU) and changes our understanding of the differential diagnosis and management [5]. So far, more than 29 patients have been described in the literature, most of them diagnosed through whole-exome sequencing [1, 6–11]. All patients tested presented with low levels of the cerebrospinal fluid (CSF) neurotransmitter metabolites homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), but with normal CSF, urinary, or blood neopterin and biopterin. They all responded to oral loading with BH4 (20 mg/kg) by lowering their blood phenylalanine levels. Substitution with BH4 and/or neurotransmitter precursors levodopa/carbidopa and 5-hydroxytryptophan (5-HTP) had beneficial effects in preventing the neurodevelopmental phenotype in patients treated before the onset of symptoms.

Biochemical and Genetic Background

DNAJC12, a type III member of the HSP40/DNAJ family, has been identified as the specific co-chaperone of PAH, TH, and TPHs 1 and 2) [1], the last two being rate-limiting steps in the biosynthesis of catecholamines (dopamine, epinephrine, and norepinephrine) and serotonin, respectively. Dysfunction of the three hydroxylases results not only in HPA (PAH deficiency) but also in neurological and neuropsychiatric impairments (TH or the TPH deficiencies).

The function of the DNAJ proteins in the quality-control machinery is proposed to be the transfer of its specific protein clients to the molecular chaperone HSC70/HSP70–HSP90 network for proper folding [12]. This complex molecular chaperone machinery is essential to maintain the proteostasis in eukaryotic cells, not only by assisting in the folding of client proteins (e.g. PAH) but also in the intertwined triage decisions that affect the removal of misfolded proteins and thus prevent toxic aggregation and cellular damage [13].

A model of the DNAJC12/HSP70 machinery in the folding process of PAH is shown in Figure 22.1, based on [12]. DNAJC12 binds to the client protein (PAH) through its peptide-binding domain (1) and interacts with the HSP70–ATP complex through the HPD motif (conserved His, Pro, Asp signature, crucial for stimulation of HSP70’s ATPase activity) in the N-terminal J domain (2). The unfolded PAH rapidly but transiently interacts with the “open” peptide-binding site (adenine nucleotide-binding cleft) of HSP70. ATP hydrolysis is stimulated by both the J domain and PAH, causing a conformational change in HSP70 that closes the cleft and stabilizes the PAH interaction. DNAJC12 then leaves the complex (3). A nucleotide exchange factor (NEF), which has a higher affinity for HSP70–ADP than HSP70–ATP, binds HSP70 (4). The ADP subsequently dissociates through the distortion of the ATP-binding domain (5), after which ATP binds to HSP70 (6). PAH is released because of its low affinity for HSP70–ATP (7). If the native state of PAH is not attained on release, DNAJC12 rebinds to exposed hydrophobic regions and the cycle begins again.

Figure 22.1 Proposed model for the interaction between co-chaperone DNAJC12, its client PAH, and the HSP70 chaperone machinery in the refolding process of PAH. For details see text. An identical model can be proposed for the interaction between DNAJC12 and TH and TPHs 1 and 2. Modified from Kampinga HH, Craig EA. The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat Rev Mol Cell Biol. 2010;11(8):579–92. Abbreviations: HSP70, heat shock 70 KDa protein; NEF, nucleotide exchange factor; PAH, phenylalanine hydroxylase; Pi, inorganic phosphate.

Beyond its function in protein refolding, DNAJC12 is responsible for protein degradation [12] (Figure 22.2). The ubiquitin-interacting motifs (UIMs) of the DNAJC12 recognize clients (e.g. PAH) that contain a monoubiquitin or polyubiquitin moiety (1). After transfer of the client to HSP70 (2), E3 ligases (such as CHIP, i.e. carboxy-terminus of HSC70 interacting protein) and the ubiquitin conjugation machinery (UBC) can associate with the HSP70–DNAJC12 complex (theoretical proposal), leading to further ubiquitylation of the bound client (3, 4). After the canonical ATP hydrolysis step (4) and NEF-mediated nucleotide exchange (5), the polyubiquitylated PAH released from HSP70 is transferred to the proteasome for degradation.

Figure 22.2 DNAJC12-targeted degradation of PAH. For details see text. Modified from Kampinga HH, Craig EA. The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat Rev Mol Cell Biol. 2010;11(8):579–92. Abbreviations: HSP70, heat shock 70 KDa protein; NEF, nucleotide exchange factor; PAH, phenylalanine hydroxylase; Pi, inorganic phosphate; UBC, ubiquitin conjugation machinery; UIM, ubiquitin-interacting motif.

Different DNAJC-family members were previously reported to be associated with Parkinson disease, parkinsonism, and neurodegenerative diseases. Mutant DNAJC5 leads to dominant adult-onset Kufs disease and parkinsonism [14], and the overexpression of DNAJC5 induces tau release in cells [15]. Also, deleterious variants in DNAJC6 were associated with recessive juvenile parkinsonism [16], and DNAJC13 variants lead to essential tremor [17] and were associated with Parkinson disease [18]. Furthermore, deletion variants of DNAJC8 [19] and DNAJC19 [20] lead to ataxia.

It has been shown in vitro that DNAJC12 variants result in reduced PAH protein expression and activity in patients’ fibroblasts [1]. At the same time, PAH variants found in PKU patients interact with DNAJC12 different from the wild-type PAH. The interaction of normal wild-type DNAJC12 with mutant PAH in cells expressing several PAH variants associated with different forms of HPA leads to severe protein instability and accelerated PAH degradation, thus supporting the role of DNAJC12 in the processing of misfolded ubiquitinated PAH by the ubiquitin-dependent proteasome/autophagy systems [21, 22].

Clinical Phenotype

Variants in DNAJC12 were recently described to lead to mild HPA (in two patients, the newborn screening blood Phe level was normal, but elevated at the time of diagnosis), central biogenic amine deficiency, intellectual disability, and movement disorders (see next section). The initial report was on six patients from four unrelated families with HPA who exhibited neurodevelopmental delay, dystonia, and a unique profile of dopamine and serotonin deficiencies without mutations in PAH or BH4 metabolism disorder-related genes [1]. Five additional patients from three unrelated Dutch and Saudi families with homozygosity and compound heterozygosity in DNAJC12 were subsequently described with a very mild neurological phenotype (autistic features and hyperactivity), with subtle dystonia in one patient [6]. Homozygous DNAJC12 null variants have also been identified in two families with early-onset parkinsonism. Both patients had mild intellectual disability, mild non-progressive motor symptoms, sustained benefit from a small dose of levodopa, and substantial worsening of symptoms after levodopa discontinuation. Neuropathology revealed no alpha-synuclein pathology, and substantia nigra depigmentation with moderate cell loss [7]. Furthermore, DNAJC12 deficiency has been elucidated in HPA cases from Spain, as well as one from Chile with no variants in PAH and excluded BH4 deficiency. Three novel nucleotide variations were identified in 11 cases. All cases presented with HPA at diagnosis and all are currently clinically asymptomatic, except one who presented with psychomotor delay and seizures. All of these cases are consuming a normal diet, and a number of them are being treated with BH4 with a favorable response [8]. Furthermore, four cases of infants with mild HPA and global developmental delay, intellectual disability, and dystonia were reported in the last few months [9–11], and we are aware of at least eight more unreported cases (personal communication). Some patients with a mild form of DNAJC12 deficiency may present without any clinical symptoms and might remain asymptomatic even in the absence of treatment, therefore questioning the need for therapy in these mildly-affected patients.

Movement Disorder Phenotype

Due to the recent identification of DNAJC12 deficiency, the natural history remains to be delineated. Regarding movement disorders, there seems to be a spectrum of severity independent from biochemical or molecular findings (no apparent genotype–phenotype correlation and no relation with blood phenylalanine and/or neurotransmitter metabolite CSF levels). This ranges from mild and subtle movement disorders [6] to severe dystonia and extrapyramidal movement disorders in early [1] or late [10, 11] childhood, and early-onset parkinsonism in children and adults [7] (Table 22.1). Clinical characteristics are however non-specific as they reflect dopamine deficiency in the central nervous system. In contrast with other neurotransmitter disorders, autonomic features such as temperature instability or lability of blood pressure or heart rate were not observed. Oculogyric crises were reported in one patient [1]. For details on the adult movement disorder phenotype, see videos in supplemental data [7].