A novel homozygous missense variant p.D339N in the PKLR gene correlates with pyruvate kinase deficiency in a Pakistani family: a case report

We characterized, both clinically and genetically, a consanguineous Pakistani family suffering from pyruvate kinase deficiency (PKD). The family belonged to a Pashtun ethnic group living in the Peshawar municipality, and consisted of three patients; the proband (III.6), her brother (III.4), and one first cousin (III.1), all born to consanguineous parents (Fig. 1). However, genetic testing was performed on the proband only. The proband is currently a 13-year-old female who was born full term to a consanguineous couple. At clinical examination, the proband experienced chronic, most likely congenital nonautoimmune hemolytic anemia at birth, and thus was recommended for transfusion. Transfusion was started regularly since she was 22 days old, with a frequency of once a month to once every 3 months. Along with transfusion, an oral supplementation of folic acid 1 mg, daily was recommended for 30 days to stabilize the patient’s hemoglobin level. The proband had an axillary temperature of 36.1 °C, peripheral pulse rate of 114 beats per minute, respiratory rate of 24 breaths per minute, systolic blood pressure of 94 mm Hg, diastolic blood pressure of 500 mm Hg, oxygen saturation of 99%, height of 94.3 cm, weight of 13.4 kg, body mass index of 15.1 kg/m², and body surface area of 0.59 m². The proband’s height and weight remained at the third percentile though her both parents were relatively tall, likely indicating a lack of expected normal physiological development in childhood. The proband’s blood group type was AB-negative. Immunization was up to date, and no known allergies were revealed upon clinical investigation. Echocardiogram (echo) was unremarkable. Examination of the musculoskeletal, neurologic, lymphatic, and integumentary systems revealed no adverse outcomes. Abdominal examination revealed hepatomegaly (palpable, 2.6 cm below the right costal margin, smooth, not tender), splenomegaly (palpable, 2 cm below left costal margin, smooth edged, not tender; spleen size 8.8 cm). Bone marrow aspirate showed a markedly decreased myeloid to erythroid (M/E) ratio, and marked hypercellular marrow particles due to hyperplasia of the erythroid elements with normal maturation. However, myeloid maturation was normal and the number of megakaryocytes were also within the normal range, thus excluding evidence of red cell aplasia, myelodysplastic syndrome, or congenital dyserythropoietic anemia (CDA). Screening for paroxysmal nocturnal hemoglobinuria (PNH) was also negative. Measurements of blood hemoglobin (Hb) and pyruvate kinase (PK) levels were extremely low at 7.6 g/dL and 12.4 U/g Hb, respectively (Table 1). Based on the clinical findings, a final diagnosis of pyruvate kinase deficiency (PKD) was confirmed in the proband.

Sanger sequencing revealed a likely pathogenic homozygous missense variant (c.1015G > A) in exon 7 of the PKLR gene, resulting in a single amino acid substitution p.[D339N] in the PK protein. The variant p.[D339N] cosegregated with PKD phenotype in the studied family (Fig. 1). For instance, the variant was present in a homozygous state in the proband while none of the clinically unaffected family members carried the variant in a homozygous state. Of the five unaffected family members who participated in this study, four were heterozygous for the variant, while one was homozygous for the wild-type allele. To the best of our knowledge, the variant c.1015G > A has never been associated with PKD phenotype nor previously reported in the ClinVar or the Human Gene Mutation Database (HGMD). The variant was present in the gnomAD database with extremely low minor allele frequency (MAF 0.00001592); however, the allele was not present in a homozygous state. Existing in silico tools and the American College of Medical Genetics and Genomics (ACMG) classified the variant as “Likely pathogenic” (Table 1). Multiple sequence alignment of the PK orthologs showed highest conservation of Asp339 residue across vertebrate species (Fig. 2), thus reflecting the importance of Asp339 residue for PK activity. To find out the effect of this mutation on the protein’s 3D structure, we modeled wild and mutant protein structures using an online method [17]. Similarly, we performed docking using MOE software to evaluate protein–ligand interaction [18]. These computational analyses revealed that wild-type PK interact with phosphoenolpyruvate through three residues including Arg116, Glu316, and Asp339. However, the mutant protein (p.[D339N]) lost its normal interactions with phosphoenolpyruvate and developed unusual interactions through Arg216 and Glu347 (Fig. 3). Altogether, our findings suggest that p.D339N mutation possibly reduces or abolishes PK enzymatic activity leading to PK deficiency in the affected people.