A 45 year old white American male of Italian descent presented with a life-long history of occult gastrointestinal blood loss, chronic anemia, iron deficiency, and frequent bouts of abdominal pain as a child and young adult. Repeated episodes of acute gross gastrointestinal bleeding late in his fourth decade and multiple episodes of small bowel perforation required five surgical interventions between 38-45 years of age. Surgical exploration of the small intestine and intra-operative endoscopy revealed multiple recurrent small intestinal ulcerations. Histologic specimens of the ileum and jejunum revealed multiple small, well-demarcated ulcers with minimal surrounding inflammation. Stenotic ileal web formations were also described proximal to several ulcers. Misoprostol (800 mcg daily), an oral prostaglandin E1 analog, had been initiated with subsequent resolution of anemia for a period of 27 months until GI bleeding recurred again.
Colonoscopy and esophagogastroduodenoscopy had been performed on multiple occasions and always had been normal. Mesenteric arteriograms, gastrin secretion and laboratory and histologic evaluation for celiac sprue and inflammatory bowel disease were all normal. He denied having had diarrhea, nausea, vomiting or constipation. He had no history of bruising or non-gastrointestinal bleeding symptoms or severe or recurrent infections.
A clear cell renal carcinoma (Furman Grade II) was resected at age 40. A brief period of mild wheezing in early childhood was reported without subsequent respiratory complaints. A shellfish allergy causing urticaria was reported. Medications included iron sulfate and a multivitamin in addition to misoprostol. Use of nonsteroidal anti-inflammatory and corticosteroid medications was specifically denied.
The patient's father had died with multiple myeloma, and varying malignancies were reported in second and third degree relatives. No renal malignancy was reported in family members. There was no family history of ulcers.
Physical examination revealed a well-developed male. Blood pressure was normal. A 2/6 systolic murmur was heard at the left sternal border. His abdomen was notable for multiple well-healed surgical scars. Physical exam was otherwise unremarkable.
An electrocardiogram and an echocardiogram were normal. Ultrasonography demonstrated normal kidneys with evidence of prior partial resection. Blood cell counts, electrolytes, renal and liver function values were normal.
Quantification of the patient's urinary prostanoid metabolites demonstrated reduced levels compared with reference values (Table 1). The patient's mother and sister did not have reduced urinary prostanoid metabolite levels.
The finding that 11d-TxB2 excretion was reduced by 84.1% led to the hypothesis that TxA2 biosynthesis by the platelet was impaired. This was assessed by measurement of TxB2 released into the serum during blood clotting, which is derived almost entirely from platelets. The patient's mean serum TxB2 was reduced by 95.9% (Table 2). This could reflect pharmacologic inhibition of platelet COX-1, COX-1 deficiency or lack of the COX-1 substrate, AA (Figure 1). A genetic deficiency of thromboxane synthase seemed unlikely due to the depressed biosynthesis of other prostanoids. To distinguish between these possibilities, the product of the platelet 12-lipoxygenase, 12-HETE, was measured in serum; 12-HETE should be unchanged or increased with pharmacologic or genetic impairment of COX-1 catalytic activity. The level of 12-HETE in the patient's serum was decreased by 97.8% (Table 2), a finding consistent with an almost complete absence of the release of AA during platelet activation. The patient's mother and sister exhibited intermediate values for platelet-derived TxB2 and 12-HETE that were below or at the lower end of the normal range.
LTE4 was below the limit of detection of 1 pg/ml in the patient's urine whereas the range of normal values was 19-60 pg/ml.
LTB4 produced by whole blood activated by calcium ionophore from the patient measured 7.2 +- 3.3 ng/ml whereas whole blood from healthy volunteers produced 243.7 +- 22.6 ng/ml LTB4 (Table 2).
COX-1 activity was assessed by measuring production of thromboxane A2 by washed platelets after incubation with [2H8] AA. Both deuterated and non-deuterated TxB2 were quantified, reflecting conversion by COX-1 of exogenous and endogenous substrate, respectively. [2H8] TxB2 levels were similar between the patient and controls, reflecting normal COX-1 activity in the patient's platelets. Platelet activation by exogenous agonists, including arachidonic acid, induces release of endogenous arachidonic acid from cellular storage pools. Notably, non-deuterated TxB2 levels from the patient's platelets were much lower than controls (0.04 vs. 0.51 ng/103 platelets; n=1), indicating an impaired release of endogenous AA.
Platelet function was assessed by monitoring platelet aggregation and dense granule secretion in response to stimulation by ADP or collagen. ADP 5muM and collagen 2 mug/ml were the lowest doses of these reagents inducing granule secretion in all controls. ADP 5 muM induced a diminished aggregation of the patient's platelets compared with controls (n=6), mean 73.0 +- 8.29 vs. 92.67 +- 11.91 maximum % aggregation (p<0.005). Collagen 2 mug/ml also induced less aggregation in the patient, inducing a mean % maximal aggregation of 61.0 +- 14.8 vs. 95.33 +- 15.2 in controls (p<0.005). Notably, ATP release, which is a measure of dense granule secretion, in response to ADP was completely absent in the patient's platelets as opposed to controls which released a mean 0.88 +- 0.33 nmol ATP (p=0.001). ATP release in response to collagen 2mug/ml also was decreased significantly in the patient vs. controls, mean 0.16 +- 0.032 vs. 1.1 +- 0.34 nmol (p=0.002). Normal aggregation and ATP release were observed in the patient's PRP with addition of AA 250 muM.
Immunobloting for cPLA2alpha in the patient's platelets detected protein of expected molecular weight but in diminished quantity (approximately 38% of control). COX-1 was detected in equal amounts in the patient and controls.
The patient's mean total PLA2 activity in sonicated platelets represented 27.2 % of the activity in controls, with mean activity 0.77 +- 0.32 vs. 2.83 +- 0.47 pmol/min/50mug protein (p<0.0005) in controls.
Three transitions encoding non-synonymous codons in the patient's cPLA2alpha alleles were found by sequencing cPLA2alpha cDNA. These included a T to C transition (c.[331T>C]) resulting in a TCT to CCT substitution encoding a Ser to Pro change at residue 111 (p.[S111P]), a G to A transition (c.[ 1454G>A]) resulting in a CGT to CAT substitution encoding an Arg to His change at residue 485 (p.[R485H]) and an A to G transition (c.[ 1952A>G]) resulting in an AAG to AGG substitution encoding a Lys to Arg change at residue 651 (p.[K651R]). The patient was heterozygous for all three transitions and no changes were found in the 3' or 5' untranslated regions. Sequencing of cPLA2alpha cDNA from the patient's mother and sister showed that the mother was heterozygous for the p.[S111P] variant but was homozygous for the p.[R485] and p.[K651] alleles. The sister was heterozygous for both the p.[R485H] and p.[K651R] variants but was homozygous for p.[S111] alleles. This inheritance pattern demonstrates that the patient was compound heterozygous with the p.[S111P] transition on one allele and both the p.[R4885H] and p.[K651R] transitions on the other allele. All of the variants detected in cDNA products were confirmed by direct sequencing of the corresponding segments of genomic DNA.