Prolidase deficiency is a rare autosomal recessive connective tissue disorder caused by mutation in the PEPD gene. Its incidence of 1–2 in 1
000 is probably underestimated due to doctors' unfamiliarity with this condition. Moreover, the frequency might be dependent on the population considered, as is often the case for recessive disorders. Our laboratory is an international centre for the molecular diagnosis of prolidase deficiency (www.orphan.net
Only 13 mutant alleles have so far been described in 22 patients (table 3). In the past 2 years, we have collected six new patients with prolidase deficiency (table 3) and carried out molecular diagnosis by full sequencing of the prolidase transcript according to the screening method established in our laboratory.23
Table 3Summary of the patients who underwent molecular characterisation
The patients characterised in this paper have different ethnic origins: the two sisters (patients A and B) are Turkish, patient F is Turkish as well, although referred to us from a Danish doctor, patients C and D are Danish and patient E is from southern Italy.
Molecular analysis of patient A showed homozygosity for a 1234G→A mutation causing the change E412K. Patient A and her asymptomatic sister patient B, who were diagnosed only on the basis of biochemical and molecular analysis, were both homozygotic for the same mutation.
Such phenotypic variability in the presence of an identical mutation in siblings is relatively frequent in genetic disorders and its molecular basis needs further investigation. The healthy brother of patients A and B did not carry this mutation, whereas the parents were, as expected, heterozygotic carriers.
We detected the mutation 1342G→A, causing the amino acid change G448R, in three different patients: C, D and F. The same molecular defect has already been reported in four other patients with prolidase deficiency, one of whom was heterozygotic, with a null mutation on the other allele22
: the other three, two of whom were brothers, were homozygotic for this mutation.22,23
The phenotype associated with the 1342G→A mutation is similar in all described patients, all of whom presented with mild to severe skin manifestation and mental retardation.
Patient F was homozygotic for the G448R mutation. He originates from the same southern Italian region (Puglia) as two other patients harbouring the same defect, and his phenotype is similar to that of these two other patients with prolidase deficiency.23
Patient C was compound heterozygotic for the G448R and the D276N mutations and patient D was compound heterozygotic for the same G448R substitution and a G278D mutation.
The occurrence of the same change at a conservative amino acid in six unrelated families favours its relevance for protein structure or function.
The 826G→A transition responsible for the D276N change has already been described in the homozygotic state in two unrelated patients,30
who presented the typical skin ulcers, suggesting that the phenotype caused by this mutation is consistent with the classical prolidase deficiency skin manifestation. Our patient did not have any ulcers yet, but this is probably due to his young age and a follow‐up is needed for a definitive answer.
The G278D mutation was reported previously in heterozygosity with R184Q in an asymptomatic patient.24
In patient D the presence of the G448R allele could be responsible for the more severe outcome.
A phylogenetic comparison shows that R184, D276, G278, E412 and G448 are highly conserved in the prolidase sequence in organisms high up in the phylogenetic tree, such as Mus musculus (BBA11685, 93% homology with Homo sapiens prolidase, NP_000276), Emericella nidulans (CAC39600, 49% homology), Suberites domuncula (CAA75231, 80% homology), Lactobacillus delbrueckii (CAB07978, 44% homology), Pseudoalteromonas haloplanktis (AAA99824, 44% homology) and Pyrococcus furiosus (AAC61259, 43% homology; fig 2).
Figure 2Alignment of amino acid sequences for prolidase enzyme from: Homo sapiens, Mus musculus, Emericella nidulans, Suberites domuncula, Lactobacillus delbrueckii, Pseudoalteromonas haloplanktis and Pyrococcus furiosus. The asterisks indicate (more ...)
Patient E carried a homozygotic 13‐bp duplication in exon 8, generating a premature stop codon after 18 amino acids from the insertion site and resulting in the absence of prolidase.
Two other mutations described in the literature, R184X and R265X, generate a premature stop codon.25,27
Both were found in homozygotic subjects and caused the synthesis of a truncated protein. In both cases the phenotype was particularly severe, suggesting that nonsense mutations are associated with more severe clinical outcome.
The structure of human prolidase, as well as the composition of its active site, is still unknown.
Recently, the complete structure of the P furiosus
) and the organisation of its active site have been described.7,31
On the basis of homology observations, four causative mutations for prolidase deficiency are judged to occur in highly conserved amino acids that have been shown to be relevant for structure or function of Pfprol
: R184Q (R122 in Pfprol
), G278D (G211 in Pfprol
) and G448R (G323 in Pfprol
) with structural functions and D276N (D209 in Pfprol
) relevant for the cofactor metal binding, which in Pyrococcus is Co32
Figure 3Position of the pathogenic mutations in the structure of Pyrococcus furiosus prolidase. The figure shows the ribbon representation of Pfprol (PDB entry 1PV9) together with the two metal ions bound to the active site (grey spheres). The (more ...)
Using the same type of comparison, E412 corresponds to the amino acid E313 in Pfprol, which has been identified as a member of the dinuclear metal centre‐active site.
The limited number of patients who were molecularly characterised does not allow prediction concerning the ethnic origin of the mutant alleles. We can only speculate about the 1342G→A allele, found in seven patients from different countries. It does seem that it has a European origin.
The identification of de novo mutations causing prolidase deficiency will help both better understanding of the relationship between function and structure, in the absence of direct structural data, and to elucidate the relationship between molecular defects and clinical outcome.
In addition, detection of mutations in patients with prolidase deficiency allows appropriate genetic counselling: carriers can easily be detected among relatives of people in whom mutations have been identified, and knowledge about the prolidase deficiency mutations segregating in a family opens possibilities for early prenatal diagnosis.