Prolidase deficiency in an infant with an incidental finding of methaemoglobinaemia
- Chern Yan Tan ,
- Easwari Kothandaraman and
- Arunabha Ghosh
- Metabolic Medicine, Royal Manchester Children's Hospital, Manchester, UK
- Correspondence to Dr Chern Yan Tan; chernyantan@doctors.org.uk
Abstract
A 4-week-old boy presented to the hospital with symptoms of diarrhoea and vomiting initially thought to be due to cow’s milk allergy. He was discharged with extensively hydrolysed formula. The patient represented with worsening of symptoms with metabolic acidosis and was screened and treated for sepsis. However, his condition deteriorated further and he developed methaemoglobinaemia. He was transferred to the high dependency unit and was given two doses of methylene blue. Further investigations were carried out, including rapid trio exome sequencing, which identified a homozygous pathogenic Peptidase D (PEPD) variant (c.978G>A, p.(Trp326*)). This was consistent with a diagnosis of prolidase deficiency.
Background
Prolidase deficiency (PD) is a rare inborn error of metabolism, characterised by recurrent infection, skin lesions, hepatosplenomegaly, dysmorphic features and learning difficulties. Prolidase enzyme is involved in the final stage of the degradation of collagen and other proline-containing proteins, including dietary proteins.1 This case report presents a case of a 4-week-old boy who was found to have PD and an incidental finding of a methaemoglobinaemia (MetHb).
Case presentation
A 4-week-old boy (patient X) was referred by the general practitioner (GP) to the hospital with a 1-week history of vomiting and loose stools. He was vomiting three to four times a day after feeds, and passing loose stools up to 12 times a day. He was initially breast fed after birth but was subsequently changed to formula milk after the vomiting started. He was born at term via normal vaginal delivery with no complications or resuscitation at birth. All the antenatal scans were reported as normal. He is the first child of a consanguineous couple; both parents are first cousins. Father is originally from Pakistan and mother is of British Pakistani origin. There is a family history of lactose intolerance and inflammatory bowel disease. On admission, he was found to have metabolic acidosis, which was felt to be most likely secondary to viral gastroenteritis, but no further investigations were undertaken at this stage to look for underlying metabolic causes. He was kept nil by mouth for 24 hours and given intravenous fluids. Feeds were then changed to extensively hydrolysed formula (EHF) with good results and he was discharged with a diagnosis of cow’s milk allergy (CMA), with plans for outpatient dietetic follow-up.
He presented again 1 week later with continued symptoms of vomiting and diarrhoea and weight loss despite EHF. He had watery greenish stools up to 10 times a day and four episodes of vomiting each day. No other symptoms were reported. Examination findings were generally unremarkable apart from some blanching spots and Mongolian blue spots on the lower back. He was noted to have some dysmorphic features including low set ears, small mouth and flat nasal bridge. The blood gas on the second admission once again showed metabolic acidosis. Sepsis was considered as a diagnosis, and a septic screen, including blood count, blood cultures, urine culture, lumbar puncture, was performed and he was started on broad-spectrum intravenous antibiotics.
His condition deteriorated on the fifth night of admission when he was found to be dusky and pale in colour, lethargic and less responsive. The fontanelle and eyes were sunken. Blood gases revealed deteriorating metabolic acidosis with MetHb >30%. He was transferred to PICU for further management.
Investigations
He was initially screened and treated as presumed sepsis on initial presentation. Due to the persistent metabolic acidosis with normal anion gap and persistent loose stools, the metabolic team was consulted and advised on the following investigations: plasma amino acid, urine organic acid and amino acid, acylcarnitine, creatine kinase and faecal chromatography. A renal ultrasound was requested in view of persistent diarrhoea with MetHb. The renal team reviewed the patient and requested a few tests to exclude an underlying renal tubular acidosis (RTA) as a cause for the normal anion gap acidosis. The genetic team has discussed the case with the lab in Exeter and was approved for a nationally funded whole exome trio analysis. Subsequently, an endoscopy was performed to investigate the persistent symptoms of diarrhoea. The full list of investigations is as follow:
Blood results
| Bone profile | Calcium 2.06 mmol/L, corrected calcium 2.44 mmol/L, phosphate 1.30 mmol/L, ALT 14 IU/L, ALP 126 u/L, albumin 18 g/L, magnesium 0.69 mmol/L |
| Blood culture | No growth |
| Ammonia | 36 umol/L (5–50) |
| Thyroid stimulating hormone | 0.30 mu/L (0.2–5.0) |
| Free T4 | 10.5 pmol/L (9–4) |
| Creatine kinase | 26 IU/L (1-475) |
| Procalcitonin | 0.32 ug/L |
| PTH | 3.0 pmol/L (1.6–6.9) |
| G6PD | Red cell G6PD normal |
| Immunoglobulin | IgG 8.28 g/L (3.9–13) IgA 1.06 g/L (0.03–0.15) IgM 0.78 g/L (0.08–0.4) |
| Copper | 11.8 umol/L (13 – 26) |
| Zinc | 11.4 umol/L (10 – 18) |
| Selenium | 0.42 umol/L (0.2–0.6) |
| Gamma glutamyltranspeptide | 75 IU/L (10–155) |
| Cytochrome 5b reductase | Normal level |
| Hb electrophoresis | Normal |
| Plasma amino acid | Normal level of alanine, tyrosine, phenylalanine, lysine, orthinine |
| Orosomucoid | 752 mg/L |
| On admission | Day 5 |
| Blood gas → pH 7.29, CO2 5.4, HCO3 19.2, Lactate 2.2, BE – 7.0, Cl 103, Anion gap 9.8 | Blood gas (pre-methylene blue) → pH 7.16, CO2 3.9, HCO3 10.3, Lactate 5.7, BE −17, Cl 109, anion gap 12.7, MetHb >30% blood gas (postmethylene blue) → pH 7.30, CO2 4.0, HCO3 19, lactate 2.0, MetHb 7% |
| FBC → Hb 120 g/L, WCC 18.6×109 /L, neutrophils 12.69×109 /L, platelets 544×109 /L | FBC → Hb 125 g/L, WCC 40.7×109 /L, neutrophils 39×109 /L, platelets 653×109 /L |
| CRP → 31 mg/L | CRP → 75 mg/L |
| U and E → Na 132 mmol/L, K 4.1 mmol/L, urea 0.9 mmol/L, creatinine 17 umol/L, chloride 103 mmol/L, bicarbonate 19.2 mmol/L | U and E → Na 132 mmol/L, K 3.7 mmol/L, urea 3.4 mmol/L, creatinine 16 umol/L, chloride 109 mmol/L, bicarbonate 10.3 mmol/L |
CSF
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Cerebrospinal fluid (CSF) culture—no growth, white cell count (WCC) 23×106/L (46% neutrophils, 15% lymphocytes), RBC <3×106 /L.
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CSF glucose—2.9 mmol/L (lab glucose: 5.1).
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CSF protein—0.29 g/L.
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Meningococcal/pneumococcal PCR—negative.
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CSF virology (enterovirus, parechovirus, herpes simplex virus DNA type 1 or 2, varicella zoster virus)—negative.
Urine
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Urine culture—no growth.
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Urine calcium:creatinine ratio—0.041.
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Urine calcium—2.33 mmol/L.
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Urine creatinine—0.5 mmol/L.
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Urine P/C ratio—273 mg/mmol (0–30).
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Urine amino acid—imidopeptiduria (the initial urine sample was reported to have some interfering substances and a repeat was asked for. When the exome report came back, the lab had a look at both the original sample and the repeat and confirmed the presence of imidopeptiduria consistent with PD).
Stool
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Stool culture—Salmonella/Shigella/Escherichia coli 0157/Campylobacter—not isolated.
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Cryptosporidium and Giardia—not detected.
Endoscopy
Upper and lower gastrointestinal (GI) endoscopy showed no histological features suggestive of inflammatory bowel disease or ulceration in the colon or upper GI tract biopsies. There was abnormal colonic mucosa with marked regenerative hyperplastic changes and increased numbers of globlet cells and mucus production. The duodenum appeared normal with some distortion of the villous architecture. The electron microscopy and histology report have ruled out microvillous inclusions and tufted enteropathy. The changes seen in the colon biopsies could be related to ongoing mucosal healing and surface regeneration and is most probably related to the instability of the mucosal gut barrier as a result of the PD mutation.
USS
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ultrasound (USS) kidney—calcification in the lower pole of the right and left kidney in keeping with nephrocalcinosis.
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Follow-up scan showed normal appearances of liver, gallbladder, spleen and both kidneys and bowel.
Echo
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Structurally normal heart, normal left ventricle (LV) function, normal aortic arch, insignificant small patent foramen ovale (PFO).
Rapid exome
The genetic test result from the rapid exome service in Exeter has identified a homozygous pathogenic Peptidase D (PEPD) variant {c.978G>A, p.(Trp326*)} consistent with a diagnosis of PD. This variant was classified as pathogenic on the basis of The American College of Medical Genetics and Genomics (ACMG) guidelines, meeting criteria pathogenic criterion (PVS1) and absence/rarity criterion (PM2); it is predicted to result in a null allele through nonsense-mediated decay (in exon 13 of 15) and is not listed in the gnomAD database of proxy normal populations. While not previously reported in the literature, a different variant in the same codon has been reported; c.977G>A(p.Trp326*) in patients with PD and a skeletal phenotype of short stature and osteopenia.2 The genetic sample was reported in 3 weeks from the date the sample was sent. The repeat analysis at the metabolic lab was consistent with the diagnosis.
Differential diagnosis
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CMA.
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Meningitis/infection.
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Food protein-induced enterocolitis syndrome (FPIES).
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RTA.
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Underlying metabolic condition.
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Cow's milk protein allergy (CMPA) was thought to be the initial diagnosis when the patient first presented with symptoms of diarrhoea and vomiting. However, an alternative diagnosis was suspected after he represented to the hospital with similar symptoms despite on EHF.
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He was initially treated with broad-spectrum antibiotics to cover for the possibility of sepsis with or without meningitis. However, this was ruled out subsequently following negative blood and CSF culture.
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FPIES—opinion from specialist allergy team concluded that symptoms and signs were not exclusive or typical of FPIES and recommended further metabolic review. As he continued to have diarrhoea despite starting on an elemental amino acid formula Neocate, FPIES was ruled out.
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RTA—Type 1 RTA was considered as a differential as the blood gas showed a metabolic acidosis with normal anion gap and a nephrocalcinosis on the ultrasound. A virtual panel of genes associated with renal tubulopathies detected no clearly pathogenic variant.
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Plasma ammonia, amino acids, urine organic/amino acids and a rapid trio exome sequencing were sent to rule out any underlying metabolic condition. This helped to clinch the diagnosis.
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Treatment
He received a total of two doses of methylene blue. The recommended dosage of methylthioninum chloride proveblue for infants 3 months old or younger and newborn infants is 0.3–0.5 mg/kg, that is, 0.06–0.1 mL/kg, given over a period of 5 min. If the methaemoglobin levels remain significantly high, another dose of the same dosage may be repeated. The dosage for paediatric population above 3 months of age is the same as the adult population, which is 1–2 mg/kg. The medication is usually given intravenously. It is recommended to monitor the blood pressure and ECG during and after the treatment. The potential adverse reactions include hypotension and cardiac arrhythmia.3 The MetHb for the patient resolved following methylene blue infusion.
Following discharge to the ward, he continued to have diarrhoea and was referred to the paediatric gastroenterology team who instituted total parenteral nutrition (TPN). He was discussed in the weekly multidisciplinary grand round, where his progression was discussed and the input from dietitian was consulted. He demonstrated good weight gain on TPN and was started on some enteral feeds with around 30 mL of Neocate. Neocate was started as it is an amino acid-based formula and hypoallergenic, so less chance of reactions while waiting for other investigations.
Outcome and follow-up
While on the ward, patient had a few episodes of high temperature. A septic screen was repeated and he was treated for possible line sepsis.
He was discharged from the hospital while he was on five nights of PN. The PN was subsequently weaned. He is currently completely off PN and eating a normal diet with Neocate long chain polyunsaturated fatty acids (LCP) and diary-free diet. He is progressing well and parents are happy with his development. He is due for a follow-up clinic review in 2 months’ time.
Discussion
PD is a rare autosomal recessive genetic condition caused by mutations in PEPD, located on chromosome 19. This codes for prolidase, an enzyme involved in the final stage of the degradation of collagen and other proline-containing proteins, including dietary proteins. Prolidase hydrolyses the peptide bond in dipeptides with C-terminal proline or hydroxyproline.1 Collagen is an important component of the extracellular matrix, which helps to reinforce and enhance connective tissues. Approximately 30% of the amino acids in collagen are proline, and it may, therefore, act as a proline reservoir.
The actual cause of PD syndrome caused by the lack of the prolidase activity has not been clearly understood. One hypothesis is that PD leads to a defect in the recycling of proline, depleting the proline pool, impairing collagen biosynthesis and, therefore, impairing skin healing.1 Alternatively, it has been suggested that dipeptides not degraded by prolidase accumulate and lead to cell death.4 Prolidase may also play a role in the regulation of the immune system, contributing to the immunological features observed in PD.5
PD is a rare disorder, affecting approximately 1 in 1 million worldwide. It is found to be more prevalent in northern Israel, and in the Arab Moslem population.4 The phenotype of PD can be highly variable, even within the same family.6 Symptoms of PD include skin lesions, recurrent infections, dysmorphic facial features, variable intellectual disability and enlargement of the liver and spleen. The severity of skin lesions is variable, ranging from a mild rash to characteristic severe skin ulceration, most frequently affecting not only the lower limbs but also described in the upper limbs and face. Dysmorphic features may include hypertelorism, high forehead, flat nose bridge and micrognathia.4 7 An association with systemic lupus erythematosus is well described. It was reported that a child who was diagnosed with PD at the age of 6 months developed symptoms of chronic diarrhoea and recurrent infections around the age 1.8 He has a mild eczema-like erythema on the knee flexures but has not so far shown any signs of the typical skin lesions associated with PD.
A case of very early-onset Crohn disease (VEO-CD) was found in a patient with PD by the whole-exome sequencing (WES). In this case, the child presented with a 2-year history of chronic diarrhoea, recurrent scrotal oedema and severe ulcers. The colonoscopy and biopsy confirmed the diagnosis of CD. Due to the dysmorphic features and in order to investigate the unusual genetic causes of VEO-CD, WES was performed, which showed a mutation in the PEPD gene.9 Although diarrhoea has been reported in a couple of cases, it is not one of the most consistent features; therefore, it was felt appropriate to proceed with endoscopy to look for other pathology that might explain his gastrointestinal symptoms. The patient’s endoscopy showed no histological features suggestive of inflammatory bowel disease or ulceration in the colon or upper GI tract biopsies.
The laboratory findings for PD are as follows7:
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Urine amino acids—imidodipeptiduria (10–30 mmol/day).
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Full blood count (FBC)—mild anaemia (Hb >10) and mild thrombocytopenia.
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LFT—elevated AST.
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Hypergammaglobulinaemia – elevated IgG and IgA.
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Hypocomplementaemia (low C3 and C4).
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Genetic testing
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Biallelic PEPD pathogenic variant
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There is currently no definitive treatment for PD, nor is there a consensus on best practice. Management is mainly supportive with involvement of a multidisciplinary team.5 7 Various approaches to the management of dermatological symptoms have been attempted, including the use of oral supplementation of ascorbic acid or proline to enhance the collagen metabolism, manganese chlorite to increase the prolidase activity and stability and the use of corticosteroids and antihistamine to reduce the immunological response.5 I Lopes et al reported a significant improvement of the skin and less severe infections in a patient who was treated with topical proline together with a hyper-proteic diet supplemented with ascorbic acid and manganese chlorite.10 For patients with chronic ulcers, topical application of proline has been reported to be beneficial.10 11 Enzyme replacement strategies have been attempted previously either by blood transfusion or allogenic haematopoietic stem cell transplantation.5 Blood transfusion was associated with an increased in erythrocyte prolidase activity in two cases, with reported possible mild improvement in ulcer healing, but no reduction in imidopeptiduria.12 13 One patient who received stem-cell transplantation showed an improved in prolidase activity but unfortunately died few months later from a secondary infection following the transplant.14 The prognosis of PD is variable ranging from skin ulcerations to partial or full amputation of toes. The majority of patients with PD experience severe morbidity and die at an early age usually due to an infection.7
MetHb is a condition in which there is an increase in the amount of haemoglobin oxidised from ferrous (2+) to ferric (3+) state. This leads to the oxidised form of haemoglobin not being able to bind to oxygen effectively and affecting oxygen delivery, which can cause varying degree of cyanosis. The level of methaemoglobin presents in the blood (between 0% and 2%) is considered as normal. The concentration level between 10% and 20% is associated with symptoms.15
MetHb is classified into two types that are congenital and acquired. There are two forms of congenital MetHb, type I and II. Type I is more common and is caused by CYB5R functional deficiencies in red blood cells. On the other hand, type II is rare but more severe and is characterised by CYB5R in all cells. Life expectancy is short in type 2 with majority of them die in infancy.16 There has been a report on a 3-hour-old infant who failed the pulse oximetry screening was found to have type 1 hereditary cytochrome b5 reductase deficiency due to a CYB5R3 gene mutation with two pathogenic variants involving guanine to adenine substitutions.17 The acquired form of MetHb can be caused by the consumption of oxidative substances, for instance, nitrites or certain types of medications.16 Examples of medications causing MetHb include chloroquine, dapsone and bupivacaine.15
It has been reported that a 6-week-old infant presented to the hospital with a 2-day history of diarrhoea, poor feeding and lethargy. The initial pH was acidotic with a methaemoglobin of 29%. Due to sibling history of G6PD deficiency, he did not receive methylene blue. The diagnosis was thought to be FPIES causing severe diarrhoea, which resulted in MetHb.18
In another report, a 4-week old presented with a 5-day history of loose stool and as found to have metabolic acidosis with a metHb of 48%. His metHb level dropped to 3% following methylene blue administration. The infant improved after intravenous hydration and was started on elemental formula. He was discharged after 4 days of inpatient stay.19
The patient presented with symptoms of diarrhoea most probably due to his underlying condition of PD, in which the enzyme prolidase responsible in the final stage of breaking down collagen and dietary protein is lacking. Although diarrhoea is not the most common feature reported in PD, this has been found in two patients. The symptoms of diarrhoea were quite severe and he eventually developed MetHb. The MetHb might have been easily missed if the blood gas result had not been analysed appropriately or run at a machine that processes metHb. Fortunately, he improved following methylene blue and subsequent investigations revealed a diagnosis of PD.
Learning points
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Methaemoglobinaemia (MetHb) could be easily missed if clinicians do not pay attention to every component of the blood gas. It is, therefore, crucial to be vigilant when interpreting blood gases.
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MetHb should be included in the differential in a child with unexplained acidosis, particularly in the young infants.
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Cyanosis happens when the MetHb is between 10% and 20% of the total haemoglobin.
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Prolidase deficiency (PD) is a rare autosomal recessive condition, characterised by recurrent infections, skin lesions, hepatosplenomegaly and dysmorphic features.
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The gastrointestinal involvement in PD is not well defined, but there have been a small number of cases associated with very early-onset inflammatory bowel disease. Although the histology report for this patient is not suggestive of inflammatory bowel disease (IBD), it is possible that the congenital diarrhoea is related to PD, and, therefore, potentially expands the phenotype of PD.
Ethics statements
Patient consent for publication
Acknowledgments
We thanks the following individual for their contribution: 1. Alistair Horman from the Willink lab. 2. Clinical genetics in Manchester and the Exeter team. 3. The gastroenterology team and the histopathologist at the Royal Manchester Children Hospital.
Footnotes
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Contributors CYT, EK and AG have all reviewed the examined the patient and came up with this idea of writing a case report to highlight this condition and the learning points as outlined in the report. CYT has contributed to this report by gathering the necessary information, carrying out the literature search, writing up the report and referencing. EK has contributed to this report by giving suggestion on how to draft the report, reviewing previous papers, editing and making changes to the report. AG has contributed to the report by reviewing previous literature papers, editing the report and helped to provide the laboratory data.
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Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
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Case reports provide a valuable learning resource for the scientific community and can indicate areas of interest for future research. They should not be used in isolation to guide treatment choices or public health policy.
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Competing interests None declared.
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Provenance and peer review Not commissioned; externally peer reviewed.
- © BMJ Publishing Group Limited 2021. No commercial re-use. See rights and permissions. Published by BMJ.
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