Page 46 - 2020_09-Haematologica-web

P. Bianchi and E. Fermo
Mutations and clinical phenotype: the genotype-phenotype correlation
The broad spectrum of clinical presentations reported in PK deficiency reflects the extensive molecular hetero- geneity,16,26,34 and the search for a correlation between the genotype and the phenotype has been the matter of study for many years.
The genotype-phenotype correlation has been investi- gated in clinical studies and by in vitro production and characterization of recombinant mutant proteins of the human enzyme,10,13,16,70 showing that patients with severe phenotypes more commonly carry nonsense mutations or missense pathogenic variants affecting the active site or stability of the PK protein.15,16,36,71
A recent analysis evaluated the genotype-phenotype correlation in the PKD NHS.35 In addition to the volume of patients/data collected (257 patients, 177 of them unrelated), analysis of this cohort had the great advan- tage of homogeneous data collection. Mutation types were classified according to previous approaches as mis- sense (M) or non-missense (NM) (including nonsense, frameshift, splicing mutations, large deletions, in-frame indels, and promoter variants); patients with NM/NM mutations were found to have a more severe phenotype, with lower hemoglobin levels after splenectomy, a high- er number of transfusions throughout their lifetime, a higher rate of iron overload, and a higher rate of splenec- tomy, when compared with patients with M/M or M/NM PKLR mutations. This categorization has some obvious limitations; in fact, although it is easy to predict the effects of a nonsense variant, because, independently of its nature, it results in protein degradation, predicting the effect of missense variants is more complex, and must take into account the effects on functional proper- ties and the stability of the mutated protein. Studies on the biochemical characterization of recombinant mutant PK enzymes have actually warned against predictions of the effects of missense mutations simply based on the location and the nature of the replaced residues; as an example, the two most frequently reported mutations p.R486W and p.R510Q both affect arginine residues located at the A/C interface, but result in substantially different effects. The p.R486W substitution leads to an enzyme with moderately altered kinetic parameters, but does not affect protein stability, whereas the p.R510Q replacement is likely to disrupt a local network of hydro- gen bonds and ultimately results in protein instability and altered allosteric responsiveness to ATP inhibi- tion.10,13
The structural architecture of the PK molecule con- tributes greatly to the heterogeneity of biochemical properties of the abnormal variants; in fact, the majority of patients with PK deficiency are compound heterozy- gous for two missense mutations, and may therefore have several different combinations of tetramers, each with distinct kinetic, allosteric and structural properties.
In addition, it is known that in patients with identical genotypes other genetic or environmental factors may affect the phenotype. This has been observed in a large number of patients homozygous for the p.R510Q mutant reported in three studies.16,34,72 In all series, vari- ability in the severity of the disease and the well-being of the patients was observed, even within the same family. Patients displayed a wide range of hemoglobin levels
(4.9-12.2 g/dL16 and 6.7-11.5 g/dL34) with a broad spedi- um of ages at diagnosis (0-56 and 0-47 years), but similar rates of splenectomy (44% and 37%).
Phenotypic variability within the same family has been confirmed by the analysis of 88 siblings from 38 fami- lies:35 with intraclass correlations ranging from 0.4-0.61; about the same degree of similarity has been found either within or between sibling clusters for hemoglobin, total bilirubin, splenectomy, and cholecystectomy.35
Finally, PK-deficient patients usually tolerate anemia well, so the decision to transfuse or treat a patient is based on how the patient feels rather than on an arbitrary hemoglobin threshold.16 This is in part justified by the increased 2,3-diphosphogylcerate level typically found in these patients; as an important regulator of the oxygen affinity of hemoglobin, 2,3-diphosphogylcerate may enhance oxygen delivery.74 Al-Samkari et al.26 reported an illustrative case: despite continued severity of anemia after splenectomy, a PK-deficient patient did not require blood transfusion, maintaining a normal social life into adulthood. Quality of life assessments, including the Functional Assessment of Chronic Illness Therapy Fatigue subscale [FACIT-F, final score of 48 (score range 0- 52)] and the Functional Assessment of Cancer Therapy [FACT-G, score of 96 (score range 0-104)], confirmed the patient’s good quality of life.
Epigenetic factors and co-inheritance
Other causes of variability of clinical expression in PK deficiency could depend on possible individual differ- ences in metabolic or proteolytic activity, which may modulate the basic effect of the mutations on ineffective erythropoiesis74,75 or differences in splenic function, and on the ability to compensate for the enzyme deficiency by overexpressing isozymes or using alternative path- ways.16 In addition, other factors, such as genetic back- ground, concomitant functional polymorphisms of other enzymes, post-translational or epigenetic modifications, and co-inheritance of other diseases may greatly con- tribute to the phenotypic heterogeneity and complica- tions.
Patients with PK deficiency usually develop secondary iron overload with a multifactorial pathogenesis, involv- ing chronic hemolysis, ineffective erythropoiesis, and transfusion therapy;76-78 HFE mutations p.C282Y and p.H63D have been proposed as additional risk factors.79,80 Similarly, the co-inheritance of the UGT1A1 TA promoter polymorphism may contribute to the occurrence of gall- stones, which are detected with increased frequency after the first decade of life in PK-deficient patients.81,82
The concomitance of PK deficiency and other heredi- tary anemias, such as glucose-6-phosphate dehydroge- nase deficiency, hemoglobinopathies, and red blood cell membrane defects, has been reported on rare occasions, with variable contributions of the different diseases to the severity of hemolysis, and should always be considered when interpreting clinical severity.82,83 The number of these reports has grown in recent years due to the increased use of NGS technologies, allowing identifica- tion of multiple disease-associated variants in patients affected by congenital hemolytic anemias and complex patterns of inheritance.25,84,30
Heterozygosity for a mutation in the PKLR gene may
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haematologica | 2020; 105(9)