Page 54 - 2020_09-Haematologica-web

H. Al-Samkari et al.
Complexity of enzymatic and genetic diagnosis of pyruvate kinase deficiency
CASE: A 24-month old boy previously diagnosed with hered- itary xerocytosis and glucose-6-phosphate dehydrogenase defi- ciency is seen for a second opinion. He has had severe transfu- sion-dependent hemolytic anemia since birth. On evaluation, PK enzyme activity is in the normal range, but with a low PK/hex- okinase ratio. There is no family history of anemia. On evalua- tion of the patient’s parents, the patient’s mother has PK enzyme activity approximately 50% of normal and a low PK/hexokinase ratio and the patent’s father has normal PK enzyme activity and PK/hexokinase ratio.
Genetic testing is performed and the patient is found to be a compound heterozygote for c.1091G>A (p.Gly364Asp) and c.1529G>A (p.Arg510Gln) mutations in exons 8 and 11 of PKLR, respectively. As these are known pathogenic mutations, the diagnosis of PKD is confirmed. PKLR analysis of the patient’s mother showed the c.1091G>A mutation. Surprisingly,
PKLR analysis of the patient’s father revealed two wildtype alle- les and no evidence of a PKLR c.1529G>A mutation (paternity was confirmed via additional testing).
Further testing was performed to identify a possible somatic mutation in PKLR in the father. Massive parallel sequencing of the region encompassing nucleotide c.1529 of PKLR was per- formed on paternal genomic DNA isolated from peripheral blood, a buccal swab, urine, and semen. Sequencing of the same region was performed on genomic DNA isolated from peripheral blood from the patient, his mother and paternal grandparents. The results of this testing are shown in Table 2.
The presence of the c.1529G>A mutation was demonstrated in DNA of all tissues tested from the father. DNA from the paternal grandparents showed absence of the c.1529G>A mutation, thereby confirming the post-zygotic origin of the c.1529G>A mutation in the father. Altogether, these results confirmed that the father is a mosaic for the c.1529G>A mutation in PKLR.
PKD should be suspected in patients with an unex-
Table 1. Our consensus approach to routine screening of the patient with pyruvate kinase deficiency.
Condition
Cholelithiasis
Viral infections
Iron overload
Osteopenia
Endocrinopathies
Pulmonaryhypertension Extramedullary
hematopoiesis
Recommended Screening
Consider screening for gallstones with interval ultrasound examinations; discuss cholecystectomy with patient if gallstones are observed
Cholecystectomy should be considered at the time of splenectomy in patients undergoing splenectomy even in the absence of gallstones due to the high rate of gallstones in patients with pyruvate kinase deficiency (both in general and following splenectomy);40 patients should be counseled on the risk of intrahepatic cholestasis
Human immunodeficiency virus and hepatitis C annually is reasonable if receiving blood transfusions in certain countries (risk varies depending on country)
Baseline parvovirus B19 serology if parvovirus serological status unknown
Non-transfused or minimally transfused patients: ferritin yearly; R2 or T2* magnetic resonance imaging of liver and heart once
in early adulthood or when ferritin >500 μg/L with follow-up interval dependent on findings
Regularly transfused patients: ferritin every 6 months (every 3 months if receiving chelation therapy); R2 or T2* magnetic resonance imaging yearly
25-hydroxy-vitamin D levels yearly; if low, replete and re-check after 8 weeks of vitamin D repletion
DEXA baseline in early adulthood, with follow-up interval dependent on findings
Thyroid-stimulating hormone, sex hormones and fructosaminea yearly; can forego screening or increase screening interval in patients with no evidence of iron overload
Echocardiogramonceaftertheageof30orpriortopregnancy;otherwiseperformonlyforconcerningsymptoms
Perform imaging only for concerning symptoms; have a high index of suspicion for paravertebral extramedullary hematopoiesis
in cases of neuropathy or unexplained pain
aFructosamine should be used instead of hemoglobin A1c for diabetes mellitus screening in patients with hemolytic anemias.
Table 2. Results of massive parallel sequencing of the region encompassing nucleotide c.1529 of PKLR on genomic DNA isolated from peripheral blood, buccal swab, urine, and semen from members of the family of the patient in the case described in “Complexity of enzymatic and genomic diagnosis in pyruvate kinase deficiency”
Individual
Patient
Mother Father
Paternal grandfather
Paternal grandmother
Sample
Peripheral blood
Peripheral blood
Peripheral blood Urine Buccal swab Semen
Peripheral blood
Peripheral blood
PKLR genotype by Sanger sequencing for nucleotide c.1529
c.1529G>A/wt wt/wt wt/wt
wt/wt
wt/wt c.1529G>A/wt
wt/wt
wt/wt
Massive parallel sequencing
PKLR genotype
for nucleotide c.1529
c.1529G>A/wt
wt/wt
c.1529G>A/wt c.1529G>A/wt c.1529G>A/wt c.1529G>A/wt
wt/wt
wt/wt
Mutated allele frequency (%)
48.6
0
5.1 10.3 16.73 21.76
0
0
wt: wildtype.
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haematologica | 2020; 105(9)