The second patient was referred to King Faisal Specialist Hospital at 2 years of age because of recurrent chest infections that required multiple hospital admissions for treatment with intravenous antibiotics. Her most recent chest infection was complicated by empyema. There was no history of chronic diarrhea. Her parents were first cousins. She has two brothers and one sister who are alive and well. Another sister died at 3 years of age from extensive chicken pox infection.
On examination, all her growth parameters were below the third percentile. She had delayed motor milestones. Speech was appropriate for age. The tonsils were absent. The remaining systemic examination was normal. Investigations showed WBCs of 4270/mm3, an absolute neutrophil count of 2540/mm3, absolute lymphocyte count 618/mm3, hemoglobin of 9.7 g/dL, and platelets of 643×103/mm3. Immunoglobulin levels showed IgG 1000 mg/dL (N), IgM 20 mg/dL (low), and IgA 14 mg/dL (normal). Antibody response titer to tetanus was 13.48 IU/mL (protective level>0.17 IU/mL). Lymphocyte subset enumeration and proliferation responses are shown in . Erythrocyte PNP enzyme activity was undetectable. Unfortunately, material for genotype analysis was not available at the time of diagnosis and the patient was lost to follow-up.
Both patients were vaccinated with BCG, OPV and MMR among other first year killed vaccines (DTP, HIB and Hep B) with no complications.
Genomic DNA was prepared from blood samples of patient 1 by standard methods. The exons and intronexon boundaries of the PNP gene were amplified in 6 separate segments spanning exon 1, exon 2, exons 3-5, exon 6 and the promoter region. The resulting PCR fragments were sequenced on an ABI 3730 DNA Analyzer (Applied Biosystems, USA).
The primers used for the polymerase chain reactions were:
PNPex6: 5'(+) TACAGGTGTGAACCACTGC
PNP 146L cDNA was generated using the wild type PNP (146P) cDNA in the pZ vector as template and the Quick Change Mutagenesis kit from Stratagene.
The primers used to introduce the mutation were:
5'(+) TTCAGTGGTCAGAACCth T TCTCAGAGGGCCCAAT
The wild type (146P) and mutant (146L) PNP cDNAs were expressed in E coli
SΦ3834 essentially as described for human adenosine deaminase.8
Human PNP activity in extracts of transformants was analyzed histochemically after electrophoresis of bacterial lysates on cellulose acetate strips as described.6
In situ staining was also performed as described for adenosine deaminase, except that inosine was used as the substrate and exogenous PNP was omitted.
Sequencing of the amplified exons and intron-exon boundaries of the PNP gene from genomic DNA from patient 1 revealed a novel missense mutation Pro146>Leu in exon 4 due to a change in the codon from CCT>CTT (). Expression in E coli of human PNP cDNA carrying either the wild type PNP sequence or the P146L mutation revealed that the mutant protein had some residual PNP activity (). The method used to assess the activity of the mutant enzyme is semiquantitative, but these results suggest that the mutation probably does not completely eliminate PNP activity.
Sequencing of PNP gene from case 1 revealed a novel missense mution Pro146>Leu in exon 4 due to a change in the codon from CCT>CTT.
Figure 2 Histochemical in situ assay for PNP activity of recombinant wild type and P146L mutant human PNP, following expression in E coli. Lanes contained approximately equal amounts of extract protein. Lane 1, untransformed E coli SΦ3834 (control). Lane (more ...)