Introduction

Cutis laxa are typically multi-systemic disorders affecting various body parts including the skeletal, cardiovascular, pulmonary, and central nervous systems. Cutis laxa manifests in several patterns of inheritance, encompassing autosomal dominant, autosomal recessive, and X-linked (Kornak et al. 2008; Guernsey et al. 2009; Reversade et al. 2009). Autosomal recessive cutis laxa type 2B (ARCL2B, MIM 612940) caused by the mutations in PYCR1, which encodes pyrroline-5-carboxylate reductase 1 (Al-Gazali et al. 2001). PYCR1 catalyzes the ultimate step of the conversion of Δ1-pyrroline-5-carboxylate (P5C) to proline, accompanied by the oxidation of NAD(P)H to NAD(P)+ (Adams and Frank 1980). ARCL2B is characterized by cutis laxa of variable severity, abnormal physical and neuropsychological, development, and skeletal malformations. Since the first case reported by Reversade et al. (Reversade et al. 2009), approximately 70 cases carrying PYCR1 mutations have been reported, with about 84% of mutations in exons 4 to 6 (Guernsey et al. 2009; Reversade et al. 2009; Kouwenberg et al. 2011; Kretz et al. 2011; Lin et al. 2011a, b; Skidmore et al. 2011; Yildirim et al. 2011; Al-Owain et al. 2012; Martinelli et al. 2012; Zampatti et al. 2012; Nouri et al. 2013; Scherrer et al. 2013; Gardeitchik et al. 2014).

Changed protein structures of mutant PYCR1, both alone and bound with the cofactor NAD(P)H and substrate analog, have been described (Meng et al. 2006). In addition, decreased catalytic efficiency of PYCR1 for 3,4-dehydro-L-proline has been indicated from the in vitro enzymatic activity testing of PYCR1 mutation. These results suggests that enzymatic dysfunction is the underlying pathological mechanism contributing to ARCL2B (Li et al. 2017).

Here, we present a typical case of cutis laxa type 2 in a Chinese individual who carries one known mutation in PYCR1. We further analyzed the protein structure and enzymatic properties of mutant PYCR1 in vitro and confirmed its pathogenicity.

Materials and methods

Ethical compliance

The present study involved the recruitment of one Chinese patient, and its implementation was granted ethically approval by the Capital Institute of Pediatrics Ethics Committee (SHERLL 2020001). The parents of the patient provided written informed consent for the dissemination of this clinical information.

Genetic testing and interpretation

The whole-exome sequencing (WES) and mutation analysis processes have been previously described (Tian et al. 2022). The functional effects of missense mutations were predicted by five algorithms (REVEL, PolyPhen-2, SIFT, CADD, and MutationTaster).

4 × 60 K array-CGH chip (Agilent Technologies, Palo Alto, CA, USA) were used to detect genomic imbalance according to previously published methods (Shen and Wu 2009). Chip data were analyzed via DNA CytoGenomics software (Agilent Technologies, Palo Alto, CA, USA).

Neurodevelopmental evaluation

The assessment of neurodevelopmental quotient (DQ) was conducted utilizing the Children Neuropsychological and Behavior Scale-Revision 2016 (CNBS-R2016) (Li et al. 2019). This evaluation encompassed five subscales: namely gross motor skill, fine motor skill, language ability, personal-social development, and adaptive behavior.

Bioinformatics analysis of PYCR1 Ala187Thr mutation

Conservation

The UniProt IDs of protein sequences used for analyzing amino acid conservation include P32322 (Human), Q922W5 (Mouse), A0A5F5Q242 (Horse), A0A5G2R6V4 (Pig), A0A6P3YNX0 (Sheep), A0A8I6AII4 (Rat), Q58DT4 (Bovin), Q6INR2 (Xenopus laevis) and A0A1D5PKK0 (Chick). The Protein sequences were aligned using JALVIEW 2.11.2.6.

Molecular modeling and structural analysis

The high-resolution X-ray crystal structure of P5CR enzyme in complexed with NADH (PDB ID: 2GR9) was obtained from the Protein Data Bank (http://www.pdb.org). The visualization of protein structure and the analysis of amino acid exchanges were conducted using the PyMOL Molecular Graphics System, Version 2.6 by Schrödinger, LLC.

Functional assay

Preparation of plasmids and site-directed mutagenesis

The full-length PYCR1 cDNA (NM_006907.4) was amplified from normal child. The Ala187Thr mutation was generated using the overlap extension PCR mutagenesis method, with the specific primers provided in Supplementary Table 1. The wild-type(WT) and mutant(Mut) full-length PYCR1 cDNA were cloned into the pHAGE plasmid using the SalI and NotI restriction enzymes. The verifications of all constructs were conducted using Sanger sequencing (Supplementary Fig. 1).

Cell culture and transfection

The HEK 293T cells were cultivated in twelve-well tissue culture plates (106 cells per well) until reaching 80% confluency. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific Inc., Suzhou, China), complemented with 10% fetal bovine serum (FBS), 4 mM glutamine, and 1% penicillin/streptomycin at 37 °C in 5% CO2. The cells were then transfected with the constructed plasmid using Lipofectamine 3000 according to the manufacturer’s instructions (Thermo Fisher Scientific Inc., Carlsbad, CA. USA). The culture medium was changed 6 h after transfection. Cells were collected 48 h after transfection.

Western blotting

Total proteins were extracted from whole cells using the RIPA lysis buffer (Sangon Biotech, Shanghai, China). Proteins were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto 0.45 μm polyvinylidene difluoride membrane (Merck Millipore Ltd., Cork, Ireland). Proteins were probed with anti-PYCR1 (1:1000, Thermo Fisher Scientific Inc. Carlsbad, CA. USA # MA5-2752) and anti-β-actin (1:5000, Biology-med, Beijing, China # 50201) antibodies, and then incubated with horse-radish peroxidase (HRP) conjugated AffiniPure Goat polyclonal anti-mouse IgG (1:5000, Biology-med, Beijing, China # K009). Signals were detected with chemiluminescent Imaging and Analysis System (Tannon, Shanghai, China). Image-Pro Plus 6.0 software was used for densitometry quantification of WB bands.

Enzyme assays

The P5CR assay kit, employing the WST-8 colorimetric method and following the manufacturer’s instructions (Comin Biotechnology Co., Ltd., Suzhou, China), was used to determine 3,4-dehydro-L-proline dehydrogenase activity in both WT and Ala187Thr Mut PYCR1. The enzyme assays were conducted in triplicate.

Statistical analysis

The Student’s t-test was used to determine the difference in exogenous PYCR1 expression and enzymatic activity between WT and Mut subjects. A significance level of P < 0.05 was deemed to be indicative of statistical significance.

Result

Clinical features

The individual under study was delivered at full gestational term via a cesarean section procedure, originating from a healthy non-consanguineous couple. There were no antenatal or neonatal complications except for his low birth (1,400 g). He exhibited loose skin, feeding difficulties, poor physical growth, and delayed development since birth, for example, he turned over at 12 months old. He was referred to our children’s hospital at the age of 22 months. Both his height (63.6 cm, < P3) and weight (7.5 kg, < P3) were delayed. Microcephaly was noted (44 cm, < P3). He showed the distinct facial features including a triangle-shaped face, progeroid appearance, micrognathia, narrowed palpebral fissures, prominent and low-set ears, large protruding earlobes, sparse hair and broad and prominent forehead (Fig. 1a-d). Moreover, other symptoms including poor eye contact, severe malnutrition, polydactyly of the right hand, adducted thumbs, finger contractures, cryptorchidism and abnormal Babinski sign was noted. Follow-up examination at 33-month-old still showed severe muscle stiffness, even not be able to sit and hold object independently. Brain MRI was normal. His older sister was in good health.

Fig. 1
figure 1

Clinical and molecular data of the patient carrying a homozygous mutation in PYCR1. (a-d) Clinical features of the affected patient. a. Typical facial features: sparse hair, elongated triangular face, prominent and deep-set years, large protruding earlobes and prominent forehead. b. Dorsa of the feet with wrinkled skin and venous prominence. c. Skin hyper-extensibility over the abdomen. d. Adducted thumb of left hand. e. Pedigrees of the family carrying PYCR1 mutation. Filled symbols designate affected individuals. Black arrows represent the proband. f. Mutation analysis showed that the affected proband carries homozygous (c.559G > A, p.Ala187Thr) mutation (circled in red) in PYCR1, and the asymptomatic parents carry same heterozygous mutation (circled in red).

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When he was 33 months old, it was noted that he experienced significant developmental delay, as evaluated by the CNBS. He obtained a general quotient score of 7, which is equivalent to a developmental age of 1.5 months. Additionally, his gross motor age was assessed to be at2-month-old, while his fine movement age was equivalent to 0.5-month-old. His adaptability age was measured as 2.5 months, his language age as 1.5 months, and his social behavior age as 1 month.

Genetic analysis

A homozygous missense mutation (c.559G > A, p. Ala187Thr) of PYCR1 (NM_006907.4) was identified in the proband. Sanger sequencing confirmed the heterozygous mutation in his asymptomatic parents (Fig. 1e-f). No other putative pathogenic mutations or copy number variations (CNVs) were identified. This mutation was not reported in gnomAD, 1000 Genomes Project, and dbSNP database. It was rated as deleterious by multiple prediction software, including REVEL (0.917), PolyPhen-2 (0.98), SIFT score (0.001), etc.

Protein defects of PYCR1 Ala187Thr mutation from structure modeling

To determine the biological evolutionary effect of Ala187Thr mutation in PYCR1, we conducted the sequence alignment of eukaryotic PYCR1 orthologues. Our analysis revealed that Ala187Thr is conserved across all selected species (Fig. 2a), suggesting a potential functional role of this allele. We further mapped the mutation Ala187Thr into the WT P5CR protein structure (PDB ID: 2GR9). It showed that a hydrophobic residue is replaced by a polar one in the mutant protein. The structure modeling showed that the hydrogen bonds net will be modified by the substitution of the methyl group of Alanine (Ala), with the hydroxyl group of Threonine (Thr), a hydrogen bond donor group. This modification might produce a misfolding in the PYCR1 protein (Fig. 2b-c).

Fig. 2
figure 2

Bioinformatics analysis. a. Sequence alignment of eukaryotic PYCR1. Sequences are named as UniProt Entry number, followed by UniProt Entry name and amino acid sequence region. Ala 187 is highlighted in a red box. The conservation scores indicates conservative property. Conserved columns are indicated by ‘*’ (score of 11), and columns with mutations where all properties are conserved are marked with a ‘+’ (score of 10). Font size of consensus sequence indicates conservative property in the consensus histogram. b. The three-dimensional structure of the PYCR1 (ribbon representation). c. The hydrogen bonds net (dotted yellow line) and corresponding structural changes in PYCR1 introduced by p.Ala187Thr.

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Enzymatic activity assay of human WT and Mut PYCR1

The expression level of exogenous PYCR1 in p.Ala187Thr Mut 293HEK cells is decreased compared to WT cells in vitro (Fig. 3a-b). Subsequently, we performed an enzymatic assay on the transfected cells. The in vitro assay showed significantly decreased 3,4-dehydro-L-proline dehydrogenase activity in Mut cells compared with WT cells (17.78 ± 0.62 vs. 8.12 ± 0.64 nmol/min/mg protein, p < 0.01, Fig. 3c and Table S2).

Fig. 3
figure 3

P5CR enzyme activity assay. a. Western blot analysis of exogenous PYCR1 expression in HEK-293T cell. NC, transfection with empty pHAGE plasmid. WT, transfection with WT pHAGE-PYCR1 plasmid. MUT, transfection with p.Ala187Thr Mut pHAGE-PYCR1 plasmid. b. Graph shows mean ± SD of relative levels quantified by band densitometry in n = 3 independent tests. Two-sided unpaired t test. c. Comparison of enzymatic activity between WT (n = 3) and MUT (n = 3). Each data bar is the average of three independent experiments, and error bars represent the SD.

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Discussion

The typical clinical features seen in PYCR1-related ARCL2B encompass growth retardation, the presence of wrinkled and/or sagging skin, distinct facial dysmorphism, delayed psychomotor development, hypotonia, and joint hyperlaxity (Guernsey et al. 2009; Reversade et al. 2009; Zampatti et al. 2012). We present a Chinese boy harboring homozygous c.559G > A mutations in PYCR1. The patient displayed characteristic symptoms of ARCL2B, which were consistent with previous reports, including wrinkled and/or sagging skin, distinct facial dysmorphism, hyperlaxity of joints, growth retardation, hypotonia, and psychomotor developmental delay. Meanwhile, some symptoms which have been previously reported in Western patients were not observed in our patient, such as hip dislocation, hernias, wormian bones, blue sclerae, corpus callosum dysgenesis, athetoid movements, cataract and/or corneal clouding, suggesting the phenotypic heterozygosity of PYCR1 mutation. Consedering diagnosis is difficult owing to a broad clinical overlap, genetic diagnostics exhibit significant crucial and useful.

Differential neurodevelopmental trajectory has been noted in patients carrying different PYCR1 mutation, for example, patients carrying the mutations in exons 4 to 6, which encode the key catalytic regions of PYCR1 (Guernsey et al. 2009; Reversade et al. 2009), showed severe intellectual disability than the mutations in exons 1 to 2 (Dimopoulou et al. 2013). In addition, patients carrying the LOF mutation only had mild intellectual disabilities (Huang et al. 2018), including one full deletion (Kretz et al. 2011), one splicing mutation (c.138 + 1G > A) (Reversade et al. 2009). The mutation of our patient, p.Ala187Thr, has been previously reported in Western populations. However, one harbored compound heterozygous mutations (p.Ala187Thr and p.Arg116fs) and the other one’s phenotype is unknow (Huang et al. 2018; Miller et al. 2020). Notably, We found that our patient harboring homozygous p.Ala187Thr mutation presented more severe neurodevelopmental delay than one harboring compound heterozygous mutations above mentioned. An assessment of our child at 33 months old revealed extremely severe neurodevelopmental delay, while the previously reported child, only exhibited mild intellectual disability at age of 4 years. It suggested p.Ala187Thr have a higher penetrance of neurodevelopmental delay phenotype than other mutations. Despite the absence of a definitive genotype-phenotype correlation, it is still suggested that LOF and missense mutations, particularly in exons 4–6, may contribute to different phenotypes through diverse mechanisms. To the best of our knowledge, the present study is the first report of this rare homozygous missense variant related to the PYCR1 gene. The present results contribute to the mutation spectrum of PYCR1 associated diseases, which will help to understand genotype-phenotype relationship in the future.

Both of 3D structure prediction and enzymatic activity assays substantiated the p.Ala187Thr caused proline dehydrogenase functional impairment. It has been observed PYCR1 expression and intracellular proline levels decline in primary skin fibroblasts of individuals with heterozygous p.Ala187Thr mutations (Huang et al. 2018). Our Western blot data was consistent with that, which supported this missense mutation also affected protein stability. Furthermore, proline levels decline could have detrimental effects on the production of collagen and elastin inside connective tissues, ultimately resulting to cutis laxa (Mohamed et al. 2011). The primary subcellular localization of PYCR1 is within mitochondria, where it plays a crucial role in the process of de novo proline biosynthesis and the consumption of NAD(P)H (Phang et al. 2008). The underlying pathogenesis of PYCR1-related ARCL2 in mitochondria has been explored. One previous study has demonstrated that the diameter of mitochondria in patient fibroblasts is smaller, indicating a role for PYCR1 in maintaining the fine structure of mitochondria (Reversade et al. 2009). Accordingly, the manifestation of cutis laxa syndrome can be attributed to the mitochondria. In future, it would be worthwhile to explore whether its role in mitochondrial function contribute to ARCL2B and provide potential direction for disease treatment.

In summary, we report the homozygous missense mutation of PYCR1 in a boy. The patient has typical clinical findings consistent with ARCL2B and extremely severe developmental delays. A combination of computational methods and in vitro enzymatic activity assay demonstrated the adverse effects of the Ala187Thr mutation on the protein function of PYCR1.