To the Editor

Prolidase deficiency (PD) is a rare autosomal recessive disorder caused by pathogenic variants in the PEPD gene, which encodes for prolidase. This enzyme is involved in the recycling and metabolism of collagen, a major component of connective tissues [1]. Altered collagen homeostasis results in the intracellular accumulation of imidodipeptides containing proline and hydroxyproline. PD affects the structure and function of collagen-rich tissues, leading to various clinical manifestations [2]. The main features include chronic cutaneous ulcers, dysmorphic facial features, developmental delay, splenomegaly, recurrent infections, and hematological abnormalities. Interstitial lung disease (ILD) is an uncommon phenotype of PD [3].

The confirmation of PD diagnosis relies on the measurement of cellular prolidase activity and genetic testing, but not all pathogenic variants of the PEPD gene have been fully identified. In this study, we report a rare case of a Chinese girl who presented with ILD as the predominant manifestation. Whole exome sequencing revealed a novel homozygous variant of the PEPD gene (c.741–13 C > A), leading to abnormal pre-mRNA splicing.

Case Report

A 2-year-11-month-old Chinese girl presented with a 6-day history of cough and a 4-day history of dyspnea and reduced exercise tolerance. Her perinatal and family histories were unremarkable, with no consanguinity. Since infancy, she had experienced delayed growth and development. There was no history of extremity ulcerations or telangiectasias. However, she had recurrent pneumonia, requiring hospitalization 5–6 times per year. A chest X-ray from a local clinic showed diffuse pulmonary lesions. Tuberculin skin test, interferon-γ release assay, and HIV antibody test were all negative. Liver and kidney function tests, as well as the autoantibody panel were normal. The patient did not respond to a 3-day course of intravenous azithromycin for community-acquired pneumonia. She was transferred to our hospital due to progressive respiratory distress.

On admission, her vital signs were as follows: body temperature 36.5℃; pulse 98 beats/min; respiratory rate 41 breaths/min; blood pressure 85/54 mmHg. Her weight was 11 kg (< 3rd percentile) and her height 79 cm (< 3rd percentile). Peripheral oxygen saturation on room air measured 88-92%. She exhibited dysmorphic features, including a prominent forehead, flat nasal bridge, wide interocular distance, and mandibular hypoplasia (Fig. 1A). Additionally, she presented with marked digital clubbing (Fig. 1B). No lower extremity ulcerations or telangiectasias were observed. The rest of the physical examination was unremarkable.

Fig. 1
figure 1

A, Dysmorphic features of a female Chinese pediatric patient with PD characterized by prominent forehead, flat nasal bridge, wide interocular distance, mandibular hypoplasia. B, Digital clubbing. C, Chest CT images at the time of admission reveal diffuse interlobular septal thickening, accompanied by cystic changes, primarily in the peripheral and subpleural regions of both lungs. D, Subsequent imaging at the 5-month follow-up demonstrates an improvement in interstitial lesions and translucency

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Laboratory tests revealed a normal white blood cell count of 6.83 × 109/L, with 53.2% neutrophils, anemia (red blood cell count of 3.17 × 1012/L; hemoglobin level of 84 g/L), and hypothyroidism (T3 0.9 nmol/L, normal range 1.4–3.8 nmol/L; FT3 2.8 pmol/L, normal range 3.7–8.5 pmol/L; FT4 12.0 pmol/L, normal range 12.3–22.8 pmol/L). There were markedly elevated levels of IgG at 16.9 g/L (normal range 5.4–13.4 g/L), IgE at 2413.0 ng/ml (normal range 0-144.0 ng/ml), B lymphocyte percentage of 60.2% (normal range 6–25%), and serum Krebs von den Lungen-6 (KL-6) at 7383 U/mL (normal range 102–460 U/ml). Arterial blood gas analysis were normal; routine blood and urine metabolic screening were both negative. A chest computed tomography (CT) scan showed diffuse interlobular septal thickening, accompanied by cystic changes, primarily in the peripheral and subpleural regions of both lungs (Fig. 1C). A brain magnetic resonance imaging scan indicated cerebral atrophy with deepened sulci. Echocardiography revealed mild tricuspid and pulmonary valve regurgitation, with no evidence of pulmonary hypertension. Whole-exome sequencing (WES) identified a homozygous variant of the PEPD gene (c.741–13 C > A), inherited from the father and mother respectively. The c.741–13 C > A variant of the PEPD gene was predicted as uncertain by three protein function prediction software tools: SIFT, PolyPhen2, and REVEL.

Splicing Study by Minigene Analysis

To validate the function of this novel variant, we conducted a minigene assay. The wild-type plasmid transcript mRNA sequence matched the expected sequence, including complete exons 10, 11, and 12. Meanwhile, in 293 T cells transfected with the c.741–13 C > A mutant plasmid, the amplification product sizes were 378 bp and 418 bp, larger than the wild-type cDNA (367 bp) (Fig. 2A). Sanger sequencing identified 11 bp and 51 bp insertions between exon 10 and exon 11 in the mutant cDNA (Fig. 2B and C). Consequently, the amino acid sequence shift in the former results in a truncated protein (p.Ser247ArgfsX78), while the latter exhibits a 17-amino acid insertion (p.Gly246_Ser247insArgAlaCysCysArgArgMetGlyPheAspHisProProGlnAlaLeuCys).

Fig. 2
figure 2

Minigene assay for the PEPD c.741–13 C > A variant and schematic diagram of the splicing pattern. (A) Agarose gel electrophoresis of the RT-PCR products of the WT and mutated minigenes of the c.741–13 C > A variant. (B) Schematic diagram of the wild-type (WT) and mutated minigene fragments. The transcribed mRNA sequence of the WT plasmid was consistent, including complete exons 10, 11 and 12. The three transcripts: r.741-1_741-11ins, r.741-1_741-51ins, and WT sequences were transcribed by the minigene plasmid. (C) Sanger sequencing chromatograms of the reverse transcription-polymerase chain reaction products of the c.741–13 C > A variant

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Diagnosis, Treatment, and Follow-Up

The patient was diagnosed with PD and started on continuous positive airway pressure therapy for 4 days, followed by nasal prong oxygen therapy at a flow rate of 2 L/min. She also received oral prednisone at a dose of 2 mg/kg per day. After a 10-day hospital stay, the patient was discharged with home oxygen therapy. At the 1-month follow-up evaluation, she showed significant improvement in exercise capacity and was able to wean off oxygen therapy. At the subsequent 5-month follow-up evaluation, her thyroid function, IgG, and IgE levels normalized. The KL-6 level decreased to 3366 U/ml. A follow-up chest CT showed an improvement in interstitial lesions and translucency (Fig. 1B). The dose of prednisone was gradually tapered. Although her KL-6 levels and chest imaging have remained unchanged since the fifth months after discharge, the frequency of respiratory infections has significantly decreased. During the 1-year follow-up, there was only one instance of COVID-19 infection, which occurred without complications. The patient is currently taking an oral dose of 7.5 mg (0.5 mg/kg.d) of prednisone per day.

Discussion

In this study, the girl was diagnosed with PD through a combination of clinical manifestation, genetic testing, and functional experiments. She presented with ILD as the predominant manifestation, accompanied by facial deformities, short stature, developmental delay, hypothyroidism, anemia, and hypergammaglobulinemia (elevated levels of IgG and IgE). Using WES and Sanger sequencing identified a novel homozygous variant of the PEPD gene (c.741–13 C > A), leading to abnormal pre-mRNA splicing, which was confirmed by a minigene assay. Specifically, two abnormal transcripts, r.741-1_741-11ins and r.741-1_741-51ins, were transcribed by a minigene plasmid expressing the c.741–13 C > A variant, thereby confirming the pathogenicity of the variant.

Since the first report by Goodman.et al. in 1968 [1], approximately 200 cases with PD have been documented worldwide [3,4,5,6,7]. The global prevalence of PD remains unknown. However, certain populations exhibit elevated carrier frequencis due to founder variants, notably among the Ohio Amish in the USA [8], as well as the Druze and Arab Muslims in Northern Israel [9]. PD is exceptionally rare among the Chinese population. Since 1989, only three sporadic cases have been reported, with genetic results available for only one of these cases [3, 10, 11].

The clinical phenotype of PD exhibits significant heterogeneity, ranging from asymptomatic cases to potentially life-threatening conditions such as respiratory insufficiency, severe hepatitis, and cardiorenal amyloidosis [6]. Notably, Rossignol et al. reported that the most frequently manifestations are dermatologic lesions (84%), followed by dysmorphic features (67%), developmental anomalies (58%), recurrent or severe infections (48%), and splenomegaly (45%) between 1968 and 2020 [6]. Dermatological lesions may not present as the first signs of PD; instead, they are part of a spectrum of symptoms that emerge gradually from the neonatal stage through to adulthood [12].

Pulmonary involvement in PD occurs in approximately 26% of cases, primarily manifesting as recurrent or severe respiratory infections and asthma [3,4,5,6,7]. ILD is the rarest pulmonary presentation, with only eleven reported cases globally, including five in Israel [13, 14] and one each in the USA [4], Portugal [15],China [3], Japan [16], and South Asia [17], with varying degrees of severity. Chest imaging findings are varied and may present as cystic changes, ground-glass opacities, bronchiectasis, reticulation, thickening along septal and peribronchovascular lines, emphysematous changes, and air trapping. While the exact mechanisms of ILD in PD remain elusive, it is associated with disrupted collagen recycling, cell death resembling necrosis, and heightened oxidative stress, which may lead to the gradual deterioration of lung tissue [13]. Cases primarily presenting with ILD but without dermatologic lesions are exceedingly rare in PD. This highlights the importance of considering PD in the differential diagnosis of ILD, regardless of the presence of dermatologic lesions. This also emphasizes the extensive clinical spectrum of PD and the potential for misdiagnosis or underdiagnosis. Given the patient’s youth, the emergence of typical dermatological lesions warrants continued observation.

Certain PD patients exhibit mutations in the PEPD gene, located at 19q13.11. According to the Human Gene Mutation Database (July 2023), there are 61 documented PEPD mutations, including 38 missense/nonsense, 7 splice site, 10 microdeletions, 2 microduplications, and 3 large deletions. Notably, a prevalent mutation, c.605 C > T, accounts for 85% (17/20) of PD cases in Israel [14]. However, the genotype-phenotype relationship remains unclear. Rossignol et al. indicate that biallelic missense variant carriers tend to develop ulcers less frequently and later than those with loss-of-function variants [6]. In our case, a child with a homozygous mutation inherited from both parents suggests an ancestral link, despite no reported consanguinity. Additionally, a novel splice site mutation, c.741–13 C > A, disrupts pre-mRNA splicing, resulting in two abnormal transcripts: r.741-1_741-11ins and r.741-1_741-51ins, as confirmed by a minigene assay, thus establishing its pathogenicity. This discovery expands the spectrum of known PEPD gene mutations.

The treatment of PD remains symptomatic, with no recommended or curative regimen. For managing cutaneous ulcers, topical and systemic treatments only provide partial temporary benefit. For autoimmunity, rituximab has shown promising efficacy [18]. Efforts to replace prolidase activity, including blood transfusions, gene therapy, and enzyme replacement, have shown limited efficacy [12]. The efficacy of hematopoietic stem cell transplantation remains controversial [19, 20]. Hyperimmunoglobulinemia E is common in PD due to the binding of gamma globulins to the prolidase substrate in serum and as a result of repeated infections and immunological dysregulation [2]. The efficacy of corticosteroids on cutaneous lesions is likely mediated by inhibiting the infiltration of polymorphonuclear leukocytes and the generation of superoxide by neutrophils [21]. However, the role of corticosteroids in patients with ILD remains unassessed. A long-term follow-up study by Cottin et al. indicated that while fibrosis may have become less pronounced over time, emphysema and cystic changes continued to progress, ultimately leading to chronic respiratory insufficiency [15]. Interestingly, our patient’s response to corticosteroid therapy diverged from Cottin et al.‘s findings. After 5 months of treatment, our patient demonstrated significant improvement in exercise capacity and a gradual decline in KL-6 levels, which was consistent with radiological recovery. Additionally, the notable reduction in respiratory infections at the 1-year follow-up indicates a satisfactory therapeutic outcome.