Biallelic PTRHD1 Frameshift Variants Associated with Intellectual Disability, Spasticity, and Parkinsonism

ABSTRACT

Background

PTRHD1 was proposed as a disease‐causing gene of intellectual disability, spasticity, and parkinsonism.

Objectives

To characterize the clinical phenotype and the molecular cause of intellectual disability in four affected individuals of a consanguineous family.

Methods

Clinical evaluation, whole‐exome sequencing, Sanger sequencing, reverse transcription polymerase chain reaction (PCR), real‐time PCR, immunoblot, and isoelectric focusing.

Results

A homozygous 28‐nucleotide frameshift deletion introducing a premature stop codon in the PTRHD1 exon 1 was identified in the four affected members. We further confirmed the apparent transcript escape of the nonsense‐mediated messenger RNA (mRNA) decay pathway. Real‐time PCR showed that mRNA expression of the mutant PTRHD1 is higher compared to the wild‐type. Western blotting and isoelectric focusing identified a truncated, but stable mutant PTRHD1 protein expressed in the patient's primary cells.

Conclusions

We provide further evidence that PTRHD1 mutations are associated with autosomal‐recessive childhood‐onset intellectual disability associated with spasticity and parkinsonism.

Peptidyl‐tRNA hydrolase domain‐containing one (PTRHD1) belongs to enzymes that hydrolyze transfer RNAs (tRNAs) from peptidyl‐tRNAs during translation. ¹ Although the bacterial homolog pth1 is well characterized, only sparse information is available on the functions of the eukaryotic protein. In a recent report, human PTRHD1 was described to bind weakly to peptidyl‐tRNA and did not function as peptidyl‐tRNA hydrolase. ² Currently, the functions of PTRHD1 remain unclear.

Lately, PTRHD1 biallelic genetic variants were described as causative of intellectual disability (ID) and early onset of parkinsonism in three different families. In an Iranian family with autosomal recessive parkinsonism and cognitive dysfunction, two affected members were found to have homozygosity for the variant (NM_001013663.2:c.155G>A:(p.[Cys52Tyr]) in the PTRHD1 gene. ³ , ⁴ The biallelic homozygous PTRHD1 variant (NM_001013663.2:c.157C>T:(p.[His53Tyr]) was also detected in another Iranian consanguineous family with two affected children that presented with early‐onset parkinsonism and ID. ⁵ Furthermore, a homozygous frameshift mutation (NM_001013663.2:c.169_196del, p.[Ala57Argfs*26]) of PTRHD1 was reported in three affected members in a family from South Africa. The affected members presented with ID and variable symptoms of juvenile‐onset parkinsonism. ⁶ However, to date, functional studies of the reported variants and studies of patients' derived cells were not performed.

We report on a large consanguineous family with four affected individuals presented with childhood ID. Whole exome sequencing identified a homozygous 28‐nucleotide frameshift deletion shared among affected individuals (PTRHD1:NM_001013663.2:c.169_196del, p.[Ala57Argfs*26]). Follow‐up Sanger sequencing confirmed the genotype–phenotype segregation. In the patients' cell lines, we showed that the mutated transcripts escape the nonsense‐mediated messenger RNA (mRNA) decay (NMD) pathway. Western blotting and isoelectric focusing identified a stable but truncated mutant PTRHD1 protein. This report further confirms the association of PTRHD1 pathogenic variants with human ID phenotype, spasticity, and early‐onset parkinsonism. Further studies are required to elicit the pathogenetic mechanisms of these mutations.

Subjects and Methods

A large consanguineous Omani family with four affected members (Fig. 1A) was enrolled in this study. Written informed consent was obtained from all participants or their guardians. The study was approved by the Medical Research Ethical Committee of Sultan Qaboos University (SQU MREC 1362).

FIG. 1.

(A) Three generations family pedigree, with consanguinity. Shaded symbols indicate affected individuals, and an arrow indicates the proband. The allelic genotype is shown. (B) Sanger sequencing confirmed the segregation of the identified variant (NM_001013663.2:c.169_196del) in the affected individuals. (C) Schematic diagram showing PTRHD1 consists of 2 exons that code for the domain PTH2. Wild‐type protein consists of 140 amino acids (blue indicates amino acids coded by exon 2). A red box or text indicates apparent amino acids. PTC, premature termination codon. (D) cDNA PCR products for PTRHD1 run in 2% agarose gel. PTRHD1 wild‐type (C1 and C2) had a product size of ~244 bp. Four patients' samples showed a smaller product. The parents (II‐1 and II‐2) who are heterozygous showed 2 bands.

DNA was extracted from the blood samples of all nine participants using a QIAamp Maxi Kit (Qiagen, Courtaboeuf, France). Whole exome sequencing was performed for the proband (Fig. 1A). Sanger sequencing was used to confirm candidate variants segregation within the family members (exome methodology and primers listed in Appendix S1).

After the filtration and segregation studies, we sought to assess the effect of the candidate causative variant (PTRHD1:p.[Ala57Argfs*26]) and assess the stability of the mutated transcripts. We performed real‐time polymerase chain reaction (RT‐PCR) on samples from the four affected patients, the parents, and positive and negative controls. GAPDH was used as an endogenous control for complementary DNA (cDNA) synthesis. Furthermore, we assessed the expression of the PTRHD1 mRNAs RT‐PCR. Primers used for the PCR amplification and the methodology are presented in the Appendix S1.

A western blot analysis was performed to examine the effect of the reported variant on protein expression and investigate possible protein instability and degradation. It was completed according to the standard procedure on whole cell lysate from the peripheral blood lymphocytes of four affected and two healthy individuals. Isoelectric focusing (IEF) was used for further resolution of the exact size of the truncated protein. This allowed for resolution on two dimensions, including the size and the isoelectric point. A detailed methodology report is provided in the Appendix S1.

Results

Clinical Evaluations

Clinical details of the proband (III‐7) and the affected siblings (III‐1, III‐3, and III‐4) are presented in Table 1. The four patients' phenotype was characterized by delayed milestones and moderate to severe ID. The disorder appears to be slowly progressive and variable; all patients presented initially with global developmental delay during early childhood and onset of pyramidal signs including spasticity and hyperreflexia, which in some worsened over time and led to an inability to walk (subject III‐4). Parkinsonism features developed during adulthood.

TABLE 1.

Summary of all patients reported with biallelic PTRHD1 mutations

	Current study				Kuipers et al 6			Khodadadi et al 5		Jaberi et al 3
	III‐1	III‐3	III‐4	III‐7	III‐1	III‐2	II‐3	II‐I	II‐II	PD‐102‐1	PD‐102‐2
Gender	Female	Female	Male	Female	Female	Female	Female	Male	Male	Male	Male
Origin	Omani				South African			Iranian		Iranian
Age at examination	34	30	26	10	26	29	44	39	37	34	30
Consanguinity	Yes				No			Yes		Yes
Variant	c.169_196del, p.[Ala57Argfs*26]				c.169_196del, p.[Ala57Argfs*26]			c.157C>T, p.[His53Tyr]		c.155G>A, p.[CYS52TYR]
Zygosity	Homozygous				Homozygous			Homozygous		Homozygous
Intellectual disability	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Age at onset (Parkinsonism)	Fourth decade (hypokinesia)	NA	NA	NA	Third decade	Fourth decade	Six years	Third decade	Third decade	Fourth decade	Third decade
Seizures	−	−	−	−	−	+	−	Febrile	ND	ND	ND
Spasticity	+	+	+	+	−	−	−
Hyperreflexia	+	+	+	+	+	−	−	+	+	+	ND
Babinski sign	+	+	+	−	−	−	−	+	+	+	ND
Other	Poor attention, anxiety, restlessness, aggression. Coarse facial features, gum hypertrophy, prognathism.	Social withdrawal, stranger anxiety, intermittent fear. Coarse facial features, gum hypertrophy, prognathism, upper limb contractures.	Anxiety, immobile, bedbound, upper limb contractures.	Poor concentration, restlessness, poor speech	Immobile, bedbound	Oriented, appropriate engagement	Expressionless	Poor concentration, anxiety, restlessness, hypersexual behavior, axonal sensorimotor polyneuropathy	Social withdrawal, poor eye contact	Low IQ (62), anxiety, hypersexuality, sleep disturbance, axonal sensorimotor polyneuropathy	Borderline IQ (75), depression, sleep disturbance, hypersexuality

Abbreviations: NA, not applicable; ND, not determined.

The proband (III‐7) was born at full term after an uneventful pregnancy and delivery with normal growth parameters. She sat at the age of 10 months and walked at the age of 20 months. At the age of 7 years, her speech consisted of <100 words and she was able to have 2–3 word sentences. She was placed in a special stream school, and had poor focus, and was subsequently diagnosed with attention deficit hyperactivity disorder (ADHD). She was not cooperative for the Stanford–Binet Intelligence Scale test. At the age of 10, a physical examination showed normal growth parameters, no facial dysmorphism, and a neurological examination showed spasticity and hyperreflexia. She did not exhibit signs of parkinsonism. The following investigations were within normal limits and included an inborn error of metabolism screen, thyroid function test, CK level, chromosomal microarray, and a brain magnetic resonance imaging (MRI) examination.

In the siblings (III‐1, III‐3, and III‐4), all had an uneventful pregnancy, delivery, and postnatal history. They presented with moderate to severe ID, and require assistance with daily living activities. Two patients (III‐1 and III‐3) also had slight coarsening of their facial features as they age and prognathism, which are features not described in the previous literature (Table 1). They developed generalized spasticity with brisk reflexes, but with the absence of clonus. All patients did not develop any features of extrapyramidal signs; apart from subject III‐3, she developed mild parkinsonism. She had normal eye saccades with no masking of the face or neck rigidity, however, she had a grade 1 rigidity in the right upper limb and both lower limbs on the unified Parkinson's disease rating scale (UPDRS). She had grade 2 bradykinesia in both upper extremities and grade 1 bradykinesia in the left lower extremity. She had no postural instability or tremor. She had no abnormal gait. She reported no classic non‐motor features of Parkinson's disease (PD) like hyposmia, autonomic dysfunction, or symptoms suggestive of rapid eye movement (REM)‐sleep behavior disorder. However, she had sleep onset insomnia in relation to a prominent obsessive–compulsive disorder (OCD), spending hours wiping the floor and washing her hands. She was not cooperative for the levodopa trial and OCD treatment. DaTscan imaging is not available. There was no history suggestive of seizures or developmental regression.

Genetic Analysis

After filtration and prioritization of the exome variants, we identified homozygosity for a variant (PTRHD1:NM_001013663.2:c.169_196del, p.[Ala57Argfs*26]) that could explain the phenotype. This variant segregated completely with the phenotype (Fig 1A,B). It is predicted to cause a frameshift mutation leading to the introduction of a premature stop codon, resulting in a truncated protein of ~81 amino acids (Fig. 1C). This variant was previously reported in one family to be associated with ID and juvenile parkinsonism. Other OMIM (https://omim.org/) genes that are known to be associated with ID and movement disorders were analyzed from the exome data, and no pathogenic variants were identified.

To investigate if this premature stop codon induced NMD mRNA degradation, RNA was extracted from lymphocytes for all four affected individuals and their parents, along with two control samples. All mRNA samples were then reverse‐transcribed into cDNAs. A 244 bp amplicon of PTRHD1 mRNA was amplified, and GAPDH was used as an internal control. The mutated transcript (size ≈214 bp) was stable, as shown in Fig. 1D, indicting an escape of the NMD pathway. This is contrary to the predicted in‐silico studies previously described. ⁶ Furthermore, using real‐time quantitative PCR (RT‐qPCR), we showed that there is a possible compensatory overexpression of the mutated transcript in the samples of the affected individuals (Figure S1).

The immunoblot for samples from the four affected individuals, parents, and 1 control revealed that mutant PTRHD1 protein did not undergo degradation (Figure S2). An anti‐PTRHD1 antibody that specifically binds to the N‐terminal of the PTRHD1 protein was used. To further characterize the truncated protein, we used IEF, which is a high‐resolution analytical method that allows for the separation of protein samples based on their isoelectric points (PI) and molecular weight (MW). The calculated PI for wild‐type and mutant PTRHD1 were 9.2 and 11.33, respectively. Control, parent, and affected patient whole cell lysates samples assessed. The mutant protein was observed to run at a PI of around 11 with MW of around 8–9 KDa as expected. On the other hand, the wild‐type protein showed a PI of 9 and MW of 15 KDa, whereas heterozygous samples showed the presence of both proteins. The experiment was repeated 3 times, and the results were consistent (Figure S3). Because of the lack of a clear and confirmed function of the PTRHD1, further functional assays were not performed.

Discussion

To date, only seven affected individuals from three families were described with biallelic PTRHD1 mutations. ⁶ All cases reported shared a consistent phenotype of delayed developmental milestones and moderate to severe ID. The onset of symptoms was present since late infancy. Furthermore, these patients developed pyramidal signs consisting of spasticity, hyperreflexia, and upgoing toes. Most of these patients developed levodopa‐responsive parkinsonism symptoms around the third and fourth decade. Intrafamilial and disease symptoms heterogeneity were noted, including the onset of parkinsonism symptoms. Other reported behavioral symptoms reported included depression, anxiety, sleep disturbances, and hypersexuality. In these three families, biallelic mutations of PTRHD1 were confirmed as likely causative and predicted to cause loss‐of‐function. However, further studies confirming PTRHD1 association with ID are lacking.

In a recent study, conducted in a cohort that consisted of 464 Taiwanese participants with young onset and familial PD; PTRHD1 mutations were not identified. They concluded that PTRHD1 gene mutations might not be associated with young‐onset or familial PD in Taiwanese populations. ⁷

In the present study, we report on four affected individuals with ID. We observed homozygosity for the same mutation (PTRHD1:NM_001013663: c.169_196del) reported previously. ⁶ The cohort of patients in our study presented with moderate to severe ID. The severity of symptoms within this family was variable (Table 1). Parkinsonism symptoms were present in most patients reported previously, with the age of onset extending between the third and fourth decade. However, in our cohort, only one subject developed symptoms in the fourth decade. Our cohort of patients might develop parkinsonism at a later age.

In summary, we provide further evidence that biallelic PTRHD1 mutations are associated with the phenotype of ID, spasticity, and parkinsonism. The intrafamilial symptoms variability seems to be a consistent finding. Further studies are required to elicit the functions of PTRHD1 and the pathomechanism of the disease.

Author Roles

(1) Research project: A. Conception, B. Organization, C. Execution; (2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; (3) Manuscript: A. Writing of the First Draft, B. Review and Critique.

G.K.: 1A, 1B, 1C, 2A, 2B, 3A

A.S.: 1B, 1C, 3B

A.Q.: 1B, 1C, 3B

T.J.: 1B, 1C, 3B

F.Z.: 1A, 1B, 3B

S.Y.: 1A, 2C, 3B

A.M.: 1A, 1B, 1C, 2A, 2C, 3A, 3B

Disclosures

Ethical Compliance Statement

Written informed consent was obtained from all participants or their guardians. The study was approved by the Medical Research Ethical Committee of Sultan Qaboos University (SQU MREC 1362). We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.

Funding Sources and Conflicts of Interest

This work was supported by His Majesty Trust Funds at the Sultan Qaboos University; study code: SR/MED/GENT/16/01. The authors declare that there are no conflicts of interest relevant to this work.

Financial Disclosures for the Previous 12 Months

The authors declare that there are no additional disclosures to report.

Supporting information

Appendix S1. Materials and Methods

Data Availability Statement

Data available on request from the authors.