First Japanese case of <scp>DNAJC12</scp> deficiency diagnosed after 18 years of follow‐up for mild hyperphenylalaninemia

Congenital hyperphenylalaninemia (HPA) is typically caused by a deficiency in phenylalanine hydroxylase (PAH) or disorders affecting the metabolism of tetrahydrobiopterin (BH4), a PAH co-factor. In 2017, DNAJC12 deficiency was identified as a novel cause of mild HPA.1 Since its discovery, investigations of patients with mild and unexplained HPA have identified this disorder in various regions worldwide.2-4 Here, we report the first Japanese case of DNAJC12 deficiency in an individual with mild HPA who had been followed for 18 years. The patient was tested positive for newborn screening (NBS), with Phe of 228 μmol/L. BH4 loading test was not performed during infancy. Pteridins and DHPR activities were normal. The patient was treated with Phe-free formula and mild protein restriction, with good control. At the age of 3, the patient was referred to our hospital. Direct sequencing of PAH at the age of 11, and targeted exome sequencing of PAH and BH4 related genes (PCBD1, PTS, GCH1, SPR, and QDPR) at the age of 13 revealed no pathogenic variants in the patient. Based on these negative genetic findings, dietary restrictions were gradually alleviated and eventually discontinued at the age of 15. Annual follow-up was continued. As the patient became familiar with high-protein foods, Phe levels gradually increased, occasionally exceeding 360 μmol/L. No movement or educational difficulties were reported. We analyzed DNAJC12 (NM_021800.3) in this patient at the age of 18 by targeted exome sequencing and identified a heterozygous variant, c.222dup (p.Tyr75LeufsTer2), which results in a frameshift and a premature stop codon. This variant was confirmed by Sanger sequencing, as shown in Figure 1a. It is not listed in dbSNP, ClinVar, ExAC, and gnomAD, and its allele frequency is extremely low (0.000033) in the Japanese population (http://jmorp.megabank.tohoku.ac.jp/). Copy number variation analysis also revealed a large deletion encompassing exon 5, which was confirmed by long-read sequencing using Nanopore®. Parental analysis, including Sanger sequencing, confirmed that the frameshift variant came from the mother, whereas the exon 5 deletion was of paternal origin. Neurotransmitter analysis of cerebrospinal fluid (CSF) revealed low levels of 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid, suggesting reduced serotonin and dopamine levels in CSF (Figure 1b). These findings were similar to those observed in BH4 deficiency. However, pteridin analysis of the blood, urine, and CSF confirmed normal BH4 metabolism (Figure 1b). Based on these findings, we concluded these variants to be pathogenic and that the biochemical abnormalities were caused by DNAJC12 deficiency. Thorough neurological examination revealed no symptoms such as dystonia, axial hypotonia, limb hypertonia, Parkinsonism, or nystagmus. The WAIS revealed an intelligence quotient of 79, indicating borderline intelligence, with discrepancies between subindices. Conner 3 testing did not confirm ADHD; however, signs of anxiety were evident. No brain MRI abnormalities were detected. A retrospective chart review revealed that plasma 5-HIAA and dopamine levels in this patient were, although in the normal range (Figure 1b), consistently lower than those in patients with classical phenylketonuria (PKU) (Figure 1c,d). Based on the significant 80% reduction in Phe levels in the BH4 1 week loading test (data not shown), BH4 administration was initiated along with oral serotonin and dopamine precursors. However, the patient could not tolerate the taste of BH4, and 5-hydroxytryptophan caused nausea and vomiting. Both medications were discontinued, and the patient is currently receiving only L-dopa. No follow-up spinal taps were performed. Although the attending physician perceived an improvement in the patient's responsiveness following the introduction of L-dopa, the patient reported no noticeable change. Conner 3 testing, performed 1 year after initiating L-dopa treatment, yielded almost identical results to those of previous assessments. Movement disorders have not appeared. DNAJC12 is a member of the DnaJ/Hsp40 protein family that functions as a co-chaperone with DnaK/Hsp70, facilitating the proper folding of aromatic amino acid hydroxylases, namely PAH, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH).1 These hydroxylases share a common co-factor, BH4, and the neurotransmitter profile and proposed treatment for DNAJC12 deficiency are consequently similar to those of BH4 deficiency. Symptoms of DNAJC12 deficiency include movement disorders such as dystonia or young-onset parkinsonism, developmental delay, intellectual disability, or neuropsychiatric problems such as ADHD or autism.5 The severity of the condition spans a wide spectrum with a significant number of asymptomatic patients.2, 3 For patients with apparent neurological symptoms, early diagnosis and prompt treatment are critical.4 In contrast, the age of onset of movement disorders associated with DNAJC12 deficiency ranges from infancy to adulthood,5 warranting lifelong follow-up. The Japanese guidelines for PKU/HPA recommend identifying the genetic background of each patient; DNAJC12 was added to the Japanese gene panel for HPA/PKU in 2023. Therefore, DNAJC12 deficiency should be diagnosed during infancy in the future. For patients presently under observation with unexplained mild HPA, investigating DNAJC12 may identify new cases. Since plasma 5-HIAA and dopamine levels in patients with classical PKU were significantly higher than those of the present case, these markers may be useful to differentiate DNAJC12 deficiency from HPA caused by PAH deficiency. E.O. collected and analyzed the data, prepared the figures, and drafted the manuscript. M.I. and I.M. reviewed and revised the manuscript. E.O., C.T., and N.A.-I. managed the patients. T.H. and H.S. performed the gene analyses. All authors have read and approved the final version of the manuscript. We thank Dr. Haruo Shintaku for performing pteridin and DHPR analyses. We thank the members of the Division of Newborn Screening, Tokyo Health Service Association, for the amino acid analysis. We thank the Department of Psychiatry at Nihon University Hospital for conducting WAIS and other psychiatric assessments. This study was partially supported by the Japan Agency for Medical Research and Development, AMED (Grant numbers JP17ek0109276, JP20ek0109482, and JP23ek0109636), MHLW Research Program on Rare and Intractable Diseases (Grant Number JPMH23FC1033), and Children and Families Agency Program, Government of Japan (Grant Number 23DA0801). The authors declare no conflict of interest. Written informed consent was obtained from the patient and their parents for the publication of the data included in this report. The data that support the findings of this study are available from the corresponding author upon reasonable request.