To the editor : Association between ENPP 1 K 173 Q and stroke in a newborn cohort of 395 Brazilian children with sickle cell anemia

Transcranial Doppler (TCD) screening and intensification therapymay reduce the risk of stroke from 11% to 1.9% in children with sickle cell anemia (SCA). However, the relatively low specificity of TCD screening in identifying individuals at risk of stroke highlights the need for prognostic tools with enhanced accuracy to enable the identification of individuals at the highest risk. Recently, Flanagan et al reported the association ofGOLGB1Y1212C (rs3732410) andENPP1 K173Q (rs1044498)mutationswith a decreased risk of stroke,whereas PON1Q192R (rs662) was associated with an increased risk. Thus, the present study was undertaken to evaluate the influence of these mutations on the risk of developing overt ischemic stroke (hereafter referred to as “stroke”) in a newborn cohort fromMinas Gerais, Brazil. Approval was obtained from the Hemominas Foundation institutional review board. Informed consent was provided according to the Declaration of Helsinki. The study included 395 hemoglobin SS-genotyped children aged 6 to 16 years who were followed-up at the Hemominas Foundation from birth until December 31, 2014. Strokewas defined as an acute neurological deficit lasting.24 hours. Diagnosis was confirmed in all children by imaging assessments. Our study also analyzed high-risk TCD as a second outcome, and this was defined as a time-averaged mean of the maximum velocity (TAMMX)$200 cm/s in the internal carotid ormiddle cerebral artery. Children who had suffered stroke as well as those for whom TCD screening was inadequate were excluded from the second analysis. Polymorphismswere detected by polymerase chain reaction/restriction fragment length polymorphism and confirmed by DNA sequencing in at least 5% of random samples. Of the 395 children, 23 (5.8%) had stroke. The frequency of stroke was null (0 of 83) for children who harbored the ENPP1 KK genotype, 6.8% (7 of 103) for those with QQ genotype, and 7.7% (16 of 209) for children with KQ genotype (P5 .037). The cumulative probability of stroke was significantly (P 5 .015) higher for children with theQQgenotype than for thosewith theKKgenotype (Figure 1A). Similar results were obtained comparing QQ/KQ genotypes as 1 group with the KK group (Figure 1B). Of the 333 children with valid TCD, 34 (10.2%) had high-risk readings. The frequency of high-risk TCD was 5.2% (4 of 77) for the KK genotype, 11.2% (19 of 169) for the KQ genotype, and 12.6% (11 of 87) for the QQ genotype. The cumulative probability of high-risk TCD was higher for children with the QQ genotype than for those with the KK genotype, but not significantly (P 5.09). The TAMMX of children with the QQ genotype was significantly higher than that of thosewith theKKgenotype (P5.018). No associationbetweenGOLGB1Y1212CorPON1Q192Rand stroke or high-risk TCD was detected (data not shown). In contrast with the results of Flanagan et al for whom ENPP1 Q173 was associated with a decreased risk of stroke, our findings showed instead that ENPP1 K173 was the “protective” variant. Our data further showed that only children with the KK genotype are “protected,” suggesting the dominance of the Q allele. The K173Q variant (frequently reported as K121Q because of an incorrect assignment at the start codon in early studies on ENPP1) is located in the second somatomedin-B-like domain, a noncatalytic ectodomain of ENPP1 that mediates protein homodimerization. The Q173 variant has been associated with insulin resistance, which is a condition that predisposes to cardiovascular events and type 2 diabetes. In addition, an analysis showed that the Q173 variant was significantly associated with increased blood pressure, which is a known independent risk factor for the development of stroke in individuals with SCA. Furthermore, the Q173 variant has been significantly associated with reduced nitric oxide (NO) synthase activity in human endothelial cells. NO lower bioavailability may be considered a possible pathogenic mechanism by which the ENPP1 K173Q modulates the risk of stroke in children with SCA. Evidence supporting this hypothesis includes that KK children had a significantly lower