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Human Molecular Genetics Pages 1411-1415  

Mutations in the [Delta]1-pyrroline 5-carboxylate dehydrogenase gene cause type II hyperprolinemia
Introduction
Results
   Northern blot and sequence analysis
   Expression studies
   ASO studies
Discussion
Materials And Methods
   Cell culture, DNA and RNA analysis
   Mutation survey
   Expression constructs
   Functional complementation and enzyme assay
   ASO hybridization
Acknowledgements
References

Mutations in the [Delta]<sup>1</sup>-pyrroline 5-carboxylate dehydrogenase gene cause type II hyperprolinemia

Mutations in the [Delta]1-pyrroline 5-carboxylate dehydrogenase gene cause type II hyperprolinemia

Michael T. Geraghty1, D. Vaughn4, A. J. Nicholson5, Wei-Wen Lin2, Gerardo Jimenez-Sanchez2, Cassandra Obie3, M. P. Flynn6, David Valle1,3 and Chien-an A. Hu1,3,*

1Institute for Genetic Medicine, Department of Pediatrics, 2Predoctoral Training Program in Human Genetics and Molecular Biology and 3Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA, 4National Center for Medical Genetics, OLHSC, Dublin, Ireland, 5OLLH, Drogheda, Ireland and 6Willow Brook, Mullingar, Ireland

Received April 22, 1998; Revised and Accepted June 16, 1998

We surveyed [Delta]1-pyrroline 5-carboxylate dehydrogenase genes from four patients with hyperprolinemia type II using RT-PCR amplification, genomic PCR amplification and direct sequencing. We found four mutant alleles, two with frameshift mutations [A7fs(-1) and G521fs(+1)] and two with missense mutations (S352L and P16L). To test the functional consequences of three of these, we expressed them in a P5CDh-deficient strain of Saccharomyces cerevisiae. In contrast to wild-type human P5CDh, yeast expressing S352L and G521fs(+1) failed to grow on proline and had no detectable P5CDh activity. The P16L allele, however, produced fully functional P5CDh and subsequent analysis suggests that it is polymorphic in the relevant (Spanish) population. Interestingly, the G521fs(+1) allele segregates in the large Irish Traveller pedigree used to define the HPII phenotype. To our knowledge, this is the first description of the molecular basis for this inborn error.

INTRODUCTION

Type II hyperprolinemia (HPII) is an inborn error of metabolism due to a deficiency of [Delta]1-pyrroline 5-carboxylate dehydrogenase (P5CDh) (1-4). This enzyme catalyzes the conversion of pyrroline 5-carboxylate (P5C), derived from proline or ornithine, to glutamate. The reaction is a component of the pathway connecting the urea and tricarboxylic acid cycles. Deficiency of P5CDh is inherited as an autosomal recessive trait and is characterized clinically by an increased incidence of seizures, most notably febrile seizures in childhood (5,6). Biochemically there is accumulation of proline and P5C in plasma, urine and cerebrospinal fluid (2-5). Recently, we reported the cloning of human P5CDh cDNAs encoding a 563 amino acid mitochondrial matrix protein with 42 and 26% identity with Saccharomyces cerevisiae and Escherichia coli P5CDhs, respectively (7). Expression of the full-length P5CDh cDNA in a P5CDh-deficient strain of S.cerevisiae confers measurable P5CDh activity and the ability to grow on proline as sole nitrogen source (7). Here we report the molecular analysis of P5CDh in four unrelated patients with HPII and the functional consequences of mutations in the P5CDh gene when expressed in a P5CDh-deficient strain of S.cerevisiae.

RESULTS

Northern blot and sequence analysis

A preliminary northern blot of fibroblast RNA from four unrelated HPII probands (HPII001-HPII004) probed with full-length human P5CDh cDNA showed a normal amount of normal sized (3.2 kb) transcript in all but HPII003 (Fig. 1). Using RT-PCR and sequencing, we surveyed the P5CDh cDNAs from these four probands for mutations. Three mutations were confirmed by sequencing amplified segments of genomic DNA and one by allele-specific oligonucleotide (ASO) hybridization of amplified segments of genomic DNA (see below). HPII001 is homozygous for deletion of G21, resulting in a frameshift mutation in codon 7, A7fs(-1) (Fig. 2A). HPII002 is a compound heterozygote with one A7fs(-1) allele (not shown) and a second allele with the missense mutation S352L and the synonymous mutation A350A in cis (Fig. 2B). HPII003 is homozygous for the missense mutation P16L in the predicted mitochondrial leader sequence of P5CDh (Fig. 2C) (7). HPII004 is homozygous for an allele with two mutations in cis: the synonymous mutation A417A (not shown) and a frameshift, G521fs(+1), caused by insertion of a T following bp +1563 (Fig. 2D).


Figure 1. Northern blot analysis of total fibroblast RNA from four HPII probands and two controls probed with full-length radiolabeled human P5CDh cDNA (top). A transcript of 3.2 kb is detected in all lanes except for HPII 003. RNA quality and quantity was monitored by probing the same blot with radiolabeled human OAT cDNA (below).

Figure 2. Direct sequence analysis of amplified cDNA or genomic DNA from four HPII probands. (A) A comparison of the genomic sequence containing the P5CDh A7fs(-1) allele with a control. The A7fs(-1) allele has a single G deletion (delG) at cDNA bp +21. (B) A comparison of the genomic sequence of a heterozygote for the P5CDh S352L allele with a control. The S352L allele has a C->T transition at cDNA bp +1055, which results in substitution of a leucine codon (TTG) for a serine codon (TCG). There is also a C->G transversion at cDNA bp +1050, which results in a synonymous mutation, A350A, in cis with the S352L mutation. Arrows indicate the heterozygous C1050G and C1055T mutations. (C) A comparison of the direct sequence of amplified cDNA from a homozygote for the P5CDh P16L allele with a control. The P16L allele has a C->T transition at cDNA bp +47, resulting in substitution of a leucine codon (CTC) for a proline codon (CCC). (D) A comparison of the direct sequence of amplified cDNA from a homozygote for the G521fs(+1) allele with a control. The G521fs(+1) allele has a single T insertion at cDNA bp +1563.

Expression studies

To test the functional consequences of S352L, G521fs(+1) and P16L, we expressed them in a strain of P5CDh-deficient yeast (7). Transformants expressing S352L and G521fs(+1) on a high copy (2µ-based) vector failed to grow on medium containing proline as sole nitrogen source and had no detectable P5CDh activity (Fig. 3). In contrast, transformants expressing the P16L allele grew normally on proline medium, suggesting that this mutation does not severely impair P5CDh function (Fig. 3). Furthermore, P5CDh activity in transformants expressing P16L was similar to that in yeast expressing wild-type human P5CDh (Fig. 3). Thus, in the heterologous yeast environment, P16L does not have a dramatic effect on the function of human P5CDh.

ASO studies

HPII004 is a member of a large, inbred pedigree of Irish nomads which contains many well-documented patients with hyperprolinemia (5,6). We used ASO analysis to confirm co-segregation of the G521fs(+1) allele with hyperprolinemia in a segment of this pedigree (Fig. 4). We also used ASO analysis to determine the frequency of the P16L allele in a control population. We amplified genomic DNA from 53 control individuals from the region of Spain to which the family of HPII003 traced their ancestry (Valencia). One of these 106 P5CDh alleles contained the P16L mutation (not shown).

DISCUSSION

There are two known inborn errors of proline degradation, HPI and HPII. Patients with HPII are distinguished from those with HPI by accumulation of not only proline, but also P5C, the next metabolite in the proline degradative pathway. These metabolic abnormalities were explained by the demonstration of a deficiency of P5CDh in HPII fibroblasts and lymphocytes (2,3). Additional investigation showed that P5CDh also catalyzes the oxidation of 3-hydroxy P5C, the second metabolite in the hydroxyproline degradative pathway (1). Subsequent biochemical studies indicated that P5CDh is a homodimer present in the mitochondrial matrix (8). The recent cloning of a cDNA encoding human P5CDh provided an opportunity to investigate the molecular basis of HPII (7). The human P5CDh cDNA encodes a 563 amino acid protein with a putative 24 amino acid N-terminal mitochondrial targeting sequence which, when cleaved, yields a mature monomer of 539 amino acids. In this work we provide the first description of P5CDh mutations responsible for HPII.

We examined P5CDh transcripts in four unrelated HPII probands. Their clinical and biochemical characteristics have been reported previously (3,5; see Materials and Methods). HPII001 is homozygous for deletion of G21, resulting in a frameshift mutation in codon 7, A7fs(-1), which results in premature termination of translation 22 codons downstream. We assumed that this mutation, which alters the C-terminal 98% of the P5CDh peptide, eliminates P5CDh activity. HPII002 is a compound heterozygote with one A7fs(-1) allele and a second allele with two mutations in cis: the synonymous mutation A350A and the non-conservative missense mutation S352L. HPII004 is homozygous for an allele with two mutations in cis: the synonymous mutation A417A and a frameshift, G521fs(+1), caused by insertion of a T following bp +1563. The latter alters a segment of P5CDh sequence (amino acids 521-524) highly conserved between S.cerevisiae, E.coli and mammalian P5CDhs and predicts the termination of translation nine codons downstream (7). Expression studies of both S352L and G521fs(+1) in a yeast strain deficient for P5CDh showed that transformants expressing either mutant allele failed to grow on medium containing proline as sole nitrogen source and had no detectable P5CDh activity (Fig. 3). These results confirm the deleterious functional consequences of these mutations.


Figure 3. Expression of mutant P5CDh alleles. (A) A comparison of the growth of yeast strains expressing the indicated mutant P5CDh alleles (HV7-9) with strains expressing vector alone (HV1), PUT2 (HV2), the yeast ortholog of P5CDh or wild-type human P5CDh (HV3, pHsP5CDhS2) on medium with either ammonium sulfate or proline as sole nitrogen source. The apparent slight growth advantage of pHsP5CDh-P16L (HV8) over pHsP5CDhS2 (HV3) on this plate was not a consistent finding. (B) The P5CDh activity of extracts of these strains. See Materials and Methods for a description of the various constructs.


Figure 4. Segregation of G521fs(+1) in a small segment of the Irish Traveller HPII pedigree by ASO analysis. The father and six children are all homozygous for the mutant allele; the unaffected child is heterozygous. The biochemical and clinical phenotypes are indicated below. nd, not done.

HPII004 is a member of a large pedigree of Irish Travellers, a distinct nomadic group within the Irish population with many HPII individuals (5). Studies of affected members of this pedigree showed a strong association of HPII with childhood febrile seizures (~70%), but not with mental handicap (6). We confirmed segregation of the G521fs(+1) allele in a segment of this inbred pedigree using ASO analysis (Fig. 4). The father (case IV.45 in ref. 3) had a plasma proline level of 2277 µM (normal 50-300 µM) and a urinary proline level of 22.1 mM (normal <0.1 mM). Six of his children also had HPII (urinary proline levels ranging from 40.2 to 2.1 mM), while the seventh had normal urinary proline excretion (<0.1 mM). We do not have a DNA sample from the mother, whom we infer to be heterozygous.

HPII003 is homozygous for the missense mutation P16L in the predicted mitochondrial leader sequence of P5CDh. Although this was the only mutation we detected in sequencing the entire P5CDh cDNA from this individual, several lines of evidence indicate that P16L is a neutral mutation. First, the P16L substitution occurs in the putative 24 amino acid mitochondrial targeting sequence of P5CDh and does not alter any of the residues or motifs necessary for proper targeting and processing of mitochondrial matrix proteins (7,9,10). Second, our expression studies indicate that this mutation has no effect on P5CDh activity in a heterologous yeast system. This result suggests that P16L has no effect on targeting, although it is theoretically possible that some consequence could be detected in a mammalian system. Third, ASO analysis of P5CDh genes in 53 control individuals (106 P5CDh genes) from the region of Spain to which the family of HPII003 traced their ancestry revealed one P16L allele. Although derived from a relatively small sample, the frequency of P16L (0.009) in this series is near the level of 0.01 set to define polymorphic alleles and is higher than that expected for alleles producing HPII. HPII has an estimated frequency of 1 in 200 000 (4), which would predict a carrier frequency of 1 in 223. Last, HPII003 fibroblasts exhibit a severe reduction in P5CDh mRNA (Fig. 1). A reduction in the steady-state levels of mRNA in fibroblasts has been associated with regulatory mutations or with mutations resulting in premature termination of translation (11). Based on these considerations, we hypothesize that the mutation(s) responsible for HPII in this individual is non-exonic and results in reduced steady-state levels of an otherwise normal P5CDh mRNA.

In conclusion, we found three pathological mutations, A7fs(-1), S352L and G521fs(+1), which account for six of the possible eight mutant P5CDh alleles in our four HPII probands. Additionally, we identified a presumably neutral missense mutation (P16L) in the mitochondrial targeting sequence of P5CDh in the fourth proband. These results confirm the primary role of P5CDh deficiency in HPII and provide molecular tools to identify and evaluate P5CDh mutations in other HPII families.

MATERIALS AND METHODS

Cell culture, DNA and RNA analysis

The HPII fibroblasts were from patients who have been described previously. HHII001 (GF), HPII002 (KK) and HPII003 (ED) are described in Valle et al. (3). HPII004 fibroblasts were established from IV.45, described in Flynn et al. (5). Fibroblast culture and nucleic acid isolation were as described (12,13). RNA blots were on GeneScreen Plus membranes (NEN) and hybridization and washing were with ExpressHyb (Clontech) as per the manufacturer's protocols. As probes we used full-length P5CDh (7) and OAT (14) cDNAs 32P-labeled by random hexamer priming (15).

Mutation survey

We reverse transcribed P5CDh using a specific 3[prime] primer (DV 1630, 3[prime]-CTC ACT GCA TGT ACG CGT AGC TCC AG) and a cDNA Cycle kit (InVitrogen). The resulting cDNA was amplified in three overlapping fragments: fragment A (bp -24 to +541), 5[prime]-GAA CAG CCC CGC TTC TAA CCC (DV1676), 3[prime]-CCA GCT CCA CCG CAT ACT TGG C (DV1178); fragment B (bp +468 to +1049), 5[prime]-CCA AGC GGA GAT TGA CGC (DV1431), 3[prime]-GCG GAA CAC TTC TGG CCA C (DV1108); fragment C (bp +920 to +1652), 5[prime]-CCT TCC CAC GCC TGG CTG GAG (DV1717), 3[prime]-TGT GTC TCC TTG ATG ACC TGC GG (DV1734). Amplification (35 cycles) was performed at 95°C for 30 s, 65°C for 30 s and 72°C for 30 s, followed by a final extension for 5 min. We sequenced the amplified fragments directly using internal primers (16).

To confirm the mutations at the structural gene level, we performed PCR amplification on either control or patient genomic DNA (100 ng) with primers corresponding to sequences flanking the mutation sites with an initial denaturation step of 6 min at 96°C, followed by 30 cycles of 1 min at 94°C, 2 min at 65°C and 2 min at 72°C. The amplified products were sequenced directly with the 5[prime] PCR primers. For the A7fs(-1) and P16L alleles, the PCR primers were DV1676 and DV2274 (intronic, 3[prime]-CCC TGA GGA ACC GGC GTT GAC C) and the sequencing primer was DV1676. For the S352L allele, the PCR primers were DV3381 (5[prime]-GAC GTG GAG AGC GTG GTG AG) and DV3384 (3[prime]-CCA CTT TGA TCC GAC TGT GCT) and the sequencing primer was DV3381.

Confirmation of the G521fs(+1) allele at the structural gene level was done by ASO hybridization (see below).

Expression constructs

The starting point for all P5CDh expression constructs was our previously described pHsP5CDhS2 plasmid (7). This construct has a chimeric insert with the promoter and 5[prime]-untranslated region (UTR) of the yeast P5CDh gene (PUT2) followed by the open reading frame and 3[prime]-UTR of our wild-type human P5CDh cDNA in the 2µ-based pSM703 E.coli/yeast shuttle vector. To generate the S352L allele, we utilized homologous recombination in yeast by amplifying a region of the cDNA from bp +1011 to +1285 with a 5[prime] mutant oligonucleotide containing both C1050G and C1055T and a 3[prime] wild-type oligonucleotide, as follows: 5[prime]-GAC CCT CCG CTC AGC CTT CGA GTA CGG TGG CCA GAA GTG TTC CGC GTG CTT, 3[prime]-CAA AGT AGC CCA CGG AGT CAT CAC. We linearized pHsP5CDhS2 with BssHII and co-transfected it and the amplified fragment into S.cerevisiae strain MB1472 (put2) (7). Homologous recombination produces colonies which grow on a uracil-free minimal glucose medium supplemented with 1% ammonium sulfate (MGA -ura) but not on a similar medium supplemented with 0.1% proline (MGP -ura). The recombinant plasmids were purified and sequenced to confirm the presence of the appropriate mutations. We used a similar strategy to produce the P16L allele. A 5[prime] mutant oligonucleotide was used in combination with a 3[prime] wild-type oligonucleotide to amplify a fragment from bp -30 to +335 using the patient cDNA as template: 5[prime]-ATA GAA GGC CAA TTC AAA TTC ACA GGA ATT ATG CTG CTG CCG GCG CCC GCG CTC CGC CGC GCC CTG CTG TCC CGC CTC TGG AC, 3[prime]-GCA GCC AGG GCA GCC TCA AT. pHsP5CDhS2 was linearized with NarI (bp +10) and co-transfected along with the amplified fragment into MB1472 as above. The presence of the appropriate mutation was confirmed by sequencing.

To produce a construct containing G521fs(+1), we utilized a cloning intermediate plasmid (pHsP5CDh-BS) which contains the entire yeast/human chimeric P5CDh minigene from pHsP5CDhS2 in pBluescript II (KS). We amplified a fragment of the P5CDh cDNA from HPII004 containing the G521fs(+1) mutation (bp +920 to +1693), digested it with BssHII (bp +1219) and MluI (bp +1676), ligated it into pHsP5CDh-BS and sequenced the construct to exclude Taq errors. We subcloned the G521fs(+1) P5CDh cDNA back into the yeast expression vector pSM703 (7).

Functional complementation and enzyme assay

We transformed S.cerevisiae strain MB1472 (put2) as described previously (7). Transformants grown on MGA -ura were replica plated on MGP -ura and were examined for the presence of the expected construct by isolation of the plasmid and sequencing relevant parts of the insert. We assayed P5CDh activity radioisotopically in total soluble proteins extracted from transformed yeast strains as described (2,7).

ASO hybridization

We synthesized two oligonucleotides complementary to the wild-type (ASO G521, 5[prime]-AGC AGC CCT TTG GG) and mutant [ASO G521fs(+1), 5[prime]-AGC AGC CCT TTT GG] sequences. Genomic DNA was amplified using the oligonucleotides 5[prime]-GCC ACA AAG GTG CTG AGG (exonic) and 3[prime]-CCC TGA GCT AGT GCA CTT GC (intronic). The oligonucleotides were end-labeled and hybridized to samples of the PCR-amplified products arrayed on a nylon membrane as described previously (17). The Valencian controls were anonymous adult individuals making blood donations and comprised an equal number of males and females.

ACKNOWLEDGEMENTS

This work was supported in part by a grant from the National Eye Institute (5RO1EY02948). We thank Marisa Muñoz and Javier García Conde for assistance in obtaining Valencian control DNA samples and Sandy Muscelli for manuscript preparation. D.V. is an Investigator in the Howard Hughes Medical Institute.

REFERENCES

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*To whom correspondence should be addressed at: PCTB802, 725 North Wolfe Street, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. Tel: +1 410 955 4260; Fax: +1 410 955 7397; Email: cahu@welchlink.welch.jhu.edu

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