Two novel mutations in DNAJC12 identified by whole‐exome sequencing in a patient with mild hyperphenylalaninemia

Abstract Background Recently hyperphenylalaninemia (HPA) caused by variants in DNAJC12 was reported and this suggested a new strategy for diagnosis. But DNAJC12‐associated HPA is a rare in Chinese population so far. Methods The clinical information and blood samples from the patient and his family members were collected and analyzed. Whole‐exome sequencing (WES) was used to identify the causative gene. Results We reported a newborn patient with HPA, having excluded the causes in common genes associated with HPA. By using whole‐exome sequencing, novel compound heterozygosity mutations in DNAJC12 were found, namely c.306C>G (p.His102Gln) and c.182delA (p.Lys61Argfs*6). Administering a diet with low phenylalanine combined with tetrahydrobiopterin and neurotransmitter precursors were shown to be effective in preventing neurodevelopmental delay for these patients. Conclusion Our finding confirms the diagnosis of DNAJC12‐associated HPA and suggests that genetic detection of DNAJC12 should be considered when newborn screening results are positive for HPA.


| INTRODUCTION
Hyperphenylalaninemia (HPA) is a common congenital disorder of amino acid metabolism, where the patient is unable to catalyze the hydroxylation of phenylalanine to tyrosine in the normal way. Such patients usually have variants in PAH (MIM: 612349) or PTS (MIM: 612719), which are the most common causes of HPA (Blau, 2016). Recently, HPA due to variants in a new gene, DNAJC12 (MIM: 606060), was reported. DNAJC12, also known as JDP1 or HPANBH4, is a member of the DnaJ/Hsp40 family which acts as a co-chaperone to regulate protein folding, transport, translational initiation, and gene expression (Lee, Hahn, Yun, Mita, & Chung, 2000). Most lines of research into DNAJC12 are focused on cancer and Parkinson's disease (De Bessa et al., 2006;Feng et al., 2019). The diagnosis of DNAJC12-associated HPA was reported for the first time in 2017 (Anikster et al., 2017). Subsequently, there were several reports of pathogenic mutations of DNAJC12 associated with HPA (Blau, Martinez, Hoffmann, & Thony, 2018;Veenma, Cordeiro, Sondheimer, & Mercimek-Andrews, 2018).
In the present study, we report the identification of novel compound heterozygosity mutations in DNAJC12 from a proband with HPA and no reported mutation was found in either PAH or PTS. In addition, this is the first report of 2 | SUBJECTS AND METHODS

| Ethics approval
All procedures in this study were approved by the Institutional Review Boards and Ethics Committees of Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region. Informed consent was obtained from participants in the study.

| Clinical data collection
The patient was from Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region. The clinical information and blood samples from the patient and his family members were collected and analyzed. The blood concentrations of phenylalanine and tyrosine were analyzed by tandem mass spectrometry or fluorescence detection. Urinary pterin analysis including the urinary concentrations of neopterin and biopterin were analyzed by high-performance liquid chromatography (HPLC). Dihydropteridine reductase (DHPR) activity was detected in dried blood spots.

| Molecular analysis
Genomic DNA was extracted from peripheral whole blood using the nucleic acid extraction system (Zeesan Biotech, China). Whole-exome sequencing (WES) for the patient and his family members was performed using Hiseq2000 (Illumina, San Diego, America). The TGex software (LifeMap Sciences, Alameda, CA) was used to annotate the selected variants which were confirmed by Sanger sequencing. The primers of DNAJC12 (NM_021800.2) were shown in Table S1. Rare variants were evaluated and classified following the ACMG/AMP standards and guidelines (Richards et al., 2015).

| Clinical characteristic of the patient
The proband was a full-term birth boy with normal height and weight. He was first found to be affected by HPA during newborn screening (NBS) and this was confirmed by further tests. Urinary concentrations of neopterin or biopterin and dried blood spot DHPR activity were normal. Detailed clinical information of patient is available in Table 1.

DNAJC12 deficiency
The patient first accepted Sanger sequencing of PAH and PTS, but there was no reported pathogenic variation detected. When he was a 1-year-old infant, he underwent WES to confirm the genetic cause of HPA. No mutations in the PAH, PTS, and other genes which were known to be associated with HPA were found. But two novel mutations in DNAJC12 (NM_021800.2) which he inherited from each parent, c.306C>G (p.His102Gln) and c.182delA (p.Lys61Argfs*6), were found. The two novel variants of DNAJC12 were predicted as damaging the function and had never been reported before in publicly available databases and local population databases. According to the ACMG standards and guidelines for the interpretation of sequence variants, these novel mutations are likely pathogenic or pathogenic (Table 2). Based on reported patients with HPA, who were diagnosed to be DNAJC12 deficient, we finally identified HPA in the proband which resulted from the compound heterozygosity mutations in DNAJC12. Detailed history of the family and sequencing results is shown in Figure 1.

| Clinical management and followup of the DNAJC12-deficient patient
The patient was placed on a phenylalanine-restricted diet as he was affected by HPA. When he was 1-year-old, the Gesell developmental assessment of the patient on adaptability, gross motor, fine motor, language, and social-emotional  (Figure 2). After diagnosis of DNAJC12 deficiency, the patient received a combined treatment of 5-hydroxytryptamine (50 mg/day), levodopa (100 mg/day), and tetrahydrobiopterin (25 mg/day). The re-examination of Gesell developmental assessment of the patient was normal at 2 years old, which was 103, 102, 98, 101, and 101 on adaptability, gross motor, fine motor, language, and socialemotional characteristics with the developmental quotients, respectively ( Figure 2). Through follow-up till 30 months, the patient did not onset obviously developmental delay ( Figure 2). proteins, including tyrosine hydroxylase (TH), peripheral tryptophan hydroxylase 1 (TPH1), and neuronal TPH2 (Anikster et al., 2017). PAH enzyme activity is reduced in the presence of DNAJC12 mutations (Anikster et al., 2017). DNAJC12 interacts with the ATPase domain of Hsp70 proteins to stimulate ATP hydrolysis through the J domain structure which spans positions 14-79 in DNAJC12 (Figure 3) (Kampinga & Craig, 2010). Also, it interacts with the aromatic amino acid hydroxylases (AAAHs) and plays important role in proper folding of PAH (Anikster et al., 2017;Jung-Kc et al., 2019). The BIOPKU (http://www.biopku.org) has reported 15 mutations of DNAJC12, which mainly are genetic null mutations (nonsense, frameshift, splice-site mutations) (Figure 3). The patient in the present study carried compound heterozygosity mutations of c.306C > G (p.His102Gln) and c.182delA (p.Lys61Argfs*6) in DNAJC12, which was inherited, respectively, from his parents who showed no symptoms of disease. The frameshift variant is located in the J domain structure, which was predicted to produce a truncated protein which would affect the normal function of DNAJC12. The missense variant had not been reported previously in publicly available databases, such as BIOPKU (http://www.biopku.org), ClinVar (https://www.ncbi.nlm.nih.gov/clinv ar/), HGMD (http://www. hgmd.cf.ac.uk/ac/valid ate.php), EXAC (http://exac.broad insti tute.org/), and so on. Multiple alignment of the two residues in DNAJC12 proteins from various mammalian species showed conserved ( Figure 3). Taken together, the compound heterozygosity mutations were considered to cause the HPA manifestations in the affected child.
As far as we know, this is the first report of patient with DNAJC12 deficiency in Guangxi Zhuang Autonomous Region. Both the variants in our study have never been reported. According to our in-house database (n = 2,757), the allele frequency is nearly 0.0002. Other reported patients with DNAJC12 deficiency were rare in the eastern Asian population. Furthermore, most of the loss-of-function variants of DNAJC12, noted in the Exome Aggregation Consortium (ExAC) database (http://exac.broad insti tute. org/), are not found in the eastern Asian population and the allele frequency is extremely low. Therefore, we suppose that the incidence of DNAJC12 associated with mild HPA is rare, especially in eastern Asian population.
Over the past decades, population newborn screening (NBS) has been shown to be critical for the early diagnosis and treatment of HPA (Hinton et al., 2016). The patient in our study was suspected of having HPA as a result of NBS. However, it should be noted that NBS results could be normal in some patients with DNAJC12 deficiency (Anikster et al., 2017). Those patients would have intellectual disability and/or behavioral problems when they are first diagnosed. Therefore, this study would suggest that a more sensitive and convenient screening system for HPA should be established. The reported patients with DNAJC12 deficiency usually receive BH4 with or without l-dopa/carbidopa/5-hydroxytryptophan treatment; as a result, BH4 supplementation not only supports in the aromatic amino acid hydroxylases (PAH, TH, and TPH) reactions, but also acts as a pharmacological chaperone that increases the stability of these enzymes similar to that occurs in BH4-responsive PKU (Anikster et al., 2017;de Sain-van der Velden, 2018). We also consider that the patient with DNAJC12 deficiency should receive BH4 treatment which can be effective for correcting the biochemical abnormalities and long-term prognosis. So early identification and clinical intervention of mild HPA can help prevent developmental delay and intellectual disability problems.
In this study, the main clinical symptom of the patient was HPA, but traditional experimental methods such as Sanger sequencing for several common genes could not provide the precise genetic diagnosis. Therefore, WES reanalysis is one of the important means for clinical diagnostic, especially for the patients have atypical clinical features. Furthermore, next-generation sequencing is rapidly expanding in its use for screening and diagnosing diseases (Narravula & Garber, 2017). We anticipate that WES or WGS will fulfill their potential to determine new genes and minimize uncertain findings so that patients receive optimal clinical treatment for their condition.

| CONCLUSIONS
In conclusion, we report on a patient with mild HPA and novel compound heterozygosity mutations in DNAJC12 found by WES analysis, which was HPA without the presence of PKU and BH4 metabolic disorders. The patient benefited from early intervention of a restricted diet and effective treatment. Therefore, early diagnosis and treatment are important in order to improve the neurodevelopmental outcome of HPA patients.