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Manifestations of disease in PKD
plained chronic Coombs-negative hemolytic anemia fol- lowing an unrevealing standard hemolytic anemia evalua- tion.23 Tests to be considered in this situation are outlined in Table 3, and our stepwise approach to the diagnosis of a patient with chronic unexplained hemolytic anemia is detailed in Figure 3. The first test to perform in cases of suspected PKD is the PK enzyme assay. While it is a useful and inexpensive screening test, PK enzyme activity assays have a number of limitations. Recent transfusion can result in falsely normal enzyme activity.17,23 PK enzyme activity is red cell age-dependent, and so cases of suspect- ed PKD with a normal PK enzyme assay should also be evaluated further via calculation of the PK/hexokinase ratio.32 This ratio relates PK activity to the activity of hexo- kinase, another red cell age-dependent enzyme used as an internal standard. A decreased PK/hexokinase ratio is sus- picious for PKD and should be investigated further with genetic testing.23 Of clinical relevance, PK enzyme activity has not been shown to correlate with disease severity.6, 11
In addition, this case demonstrates the complexity of genetic diagnosis in PKD. Over 300 functional mutations in PKLR have been described.33,34 While most cases can be diagnosed with standard PKLR exon sequencing of the patient, occasionally more comprehensive analysis are necessary to confirm the diagnosis or allow for family planning in subsequent pregnancies. This includes screen- ing for parental somatic mutations in cases in which an apparent de novo mutation is identified.35 In more routine cases, in which a couple is planning to conceive and one partner is a confirmed carrier of PKD (as occurs in all chil- dren of affected individuals), testing of the partner with an unconfirmed PKLR mutation status should be through sequencing rather than testing PK enzyme activity, as many carriers will have normal PK activity. An analysis of PK enzyme activity in 31 family studies (mother, father, and patient) performed by three of the authors (RvW, PB,
MMP, unpublished observation) revealed eight cases in which a carrier had normal PK activity. Therefore, in this setting enzymatic testing alone to identify possible carri- ers is inadequate and genetic testing is advised.
Occult mutations in pyruvate kinase deficiency
CASE: A 47-year old woman with PKD on regular transfu- sions following splenectomy and cholecystectomy is referred for confirmation of her diagnosis after genetic testing revealed only heterozygosity for the c.460G>A (p.Ala154Thr) mutation. She was initially diagnosed with PKD over 30 years ago and despite splenectomy in childhood has required regular transfusions (2 units every 3 weeks). PK enzyme activity measured immediately prior to transfusion is 2.5 U/g hemoglobin (reference range, 6.7- 14.3 U/g hemoglobin). The c.460G>A mutation was inherited from the patient’s mother who had normal PK activity (8.1 U/g hemoglobin) and a normal PK/hexokinase ratio of 9.0. No other relatives are available for analysis. To exclude the possibility of other causes of hereditary hemolytic anemia, a targeted next-gen- eration sequencing gene panel analysis was performed, which did not identify additional mutations in 46 genes associated with hereditary hemolytic anemias. Given the severity of her disease, she was interested in considering a clinical trial of PKD-directed treatment but was excluded because she had only one mutated PKLR allele.
While it is important to confirm an enzymatic diagnosis of PKD with genetic testing,23 up to 10% of patients with a diagnosis of PKD will have only one mutation identified on standard PKLR exome sequencing.36 It is recognized that PKLR intron splicing-associated mutations are some- times found in patients with single or no PKLR coding mutations.37 In one study of 13 kindreds with hereditary non-spherocytic hemolytic anemia and either single or no identified mutations in PKLR coding regions, whole genome sequencing identified five kindreds with unique PKLR deep intronic mutations predicted to perturb normal
Table 3. Advanced diagnostic workup of a patient with unidentified Coombs-negative hemolytic anemia to be considered after basic testing (peripheral blood film, hemoglobin electrophoresis, etc.). Often many of these tests will be indicated in such a patient and may be obtained in a stepwise fashion to diagnose the underlying disorder. Our consensus stepwise approach is given in Figure 1.
Test
Pyruvate kinase enzyme assay, other glycolytic enzyme testing, pyrimidine 5’-nucleotidase testing
Extended direct antiglobulin testing
(e.g. IgA detection, elution)
Osmotic gradient ektacytometry, osmotic fragility test, eosin-5-maleimide binding, genetic testing for red cell membrane mutations
Flow cytometry for CD55 and CD59, multiparameter fluorescent aerolysin-based flow cytometry
Ceruloplasmin
ADAMTS13 activity
Genetic testing for unstable hemoglobin variants
Targeted next-generation sequencing
panels
Situations to Consider
Chronic/lifelong hemolytic anemia with bland peripheral blood film, possible splenomegaly; certain glycolytic defects may present with other pathological features (e.g. severe neuromuscular symptoms in triosephosphate isomerase deficiency, myopathy in phosphofructokinase deficiency)
Basophilic stippling of red cells common in pyrimidine 5’-nucleotidase deficiency
Evidence of autoimmune hemolysis (e.g. spherocytes, microspherocytes) but standard direct antiglobulin
test negative for IgG and C3
Possible red cell membrane disorders (e.g. hereditary spherocytosis, hereditary elliptocytosis, hereditary xerocytosis) or congenital dyserythropoietic anemias
Hemolysis with thrombosis and/or other cytopenias
Screening for Wilson disease in a patient with unidentified Coombs-negative hemolytic anemia and psychiatric/neurological symptoms, evidence of liver dysfunction, and/or Kayser-Fleischer rings
Concomitant thrombocytopenia in a patient with chronic unidentified hemolysis is concerning for congenital
thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome)
Chronic/lifelong hemolytic anemia with bland peripheral blood film or basophilic stippling of red cells, possible splenomegaly
Cases with unexplained hemolytic anemia after comprehensive hematologic testing or when hematologic
testing cannot be performed accurately (i.e. in recently-transfused patients, neonates, or shipped samples)
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