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Genetic Prothrombotic Mutations Are Common in Neonates but Are Not Associated with Umbilical Catheter-Associated Thrombosis

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Genetic Prothrombotic Mutations Are Common in Neonates but Are Not Associated with Umbilical Catheter-Associated Thrombosis Journal of Perinatology (2007) 27, 490–495 r 2007 Nature Publishing Group All rights reserved. 0743-8346/07 $30 www.nature.com/jp ORIGINAL ARTICLE Genetic prothrombotic mutations are common in neonates but are not associated with umbilical catheter-associated thrombosis R Turebylu1, R Salis2, R Erbe2, D Martin3, S Lakshminrusimha1 and RM Ryan1,4,5 1Division of Neonatology, Department of Pediatrics, State University of New York at Buffalo, Women and Children’s Hospital of Buffalo, Buffalo, NY, USA; 2Division of Genetics, Department of Pediatrics, State University of New York at Buffalo, Women and Children’s Hospital of Buffalo, Buffalo, NY, USA; 3Department of Radiology, State University of New York at Buffalo, Women and Children’s Hospital of Buffalo, Buffalo, NY, USA; 4Department of Pathology and Anatomical Sciences, State University of New York at Buffalo, Women and Children’s Hospital of Buffalo, Buffalo, NY, USA and 5Department of Gynecology-Obstetrics, State University of New York at Buffalo, Women and Children’s Hospital of Buffalo, Buffalo, NY, USA Keywords: umbilical catheter; thrombosis; duplex Doppler ultrasound; Objective: To evaluate the prevalence of hereditary prothrombotic factor V Leiden; methylene-tetrahydrofolate reductase mutation; mutations, and their effect on the incidence and severity of umbilical prothrombin 20210 G>A mutation arterial or venous catheter (UAC or UVC)-associated thrombosis. Study Design: All neonates with a UAC or UVC were studied prospectively for the presence, severity and timing of thrombosis with Introduction duplex Doppler ultrasound scan. Genetic testing for factor V Leiden (FVL), Neonatal umbilical arterial catheter (UAC) and umbilical venous prothrombin mutation (PTm) and methylene-tetrahydrofolate reductase catheter (UVC) usage is routine practice in many neonatal (MTHFR) mutations was performed using PCR and restriction fragment intensive care units (NICUs). UAC usage has been reported in length polymorphism assays. 10–64% of all NICU admissions.1 Thrombosis as a complication of 2–5 Result: Umbilical catheter (UC)-associated thrombosis developed in UAC use is common, with an incidence range of 4.5 to 90%. 16/53 (31%) neonates; 23% of UACs and 22% of UVCs were associated with Complications of aortic thrombosis include gut ischemia, thrombosis. The prevalence of a significant prothrombotic mutation was hypertension, decreased renal perfusion, complete aortic occlusion 6–9 present in 10/51 (20%) of infants: FVL (8%), MTHFR667 homozygosity and congestive heart failure. Thrombosis complicating UVC use 4,10,11 (10%), MTHFR1298 homozygosity (2%) and PTm (0%). There was no is also common, with a reported incidence of 4.1 to 44%. increase in the risk of UC-associated thrombus in patients carrying these There has been a recent flourish in the identification of genetic prothrombotic mutations; our study had the power to detect a 2.5-fold abnormalities associated with increased thrombotic risk. Genetic increased risk of thrombosis for any of these significant mutations. In prothrombotic factors are more prevalent in patients with addition, MTHFR667 heterozygosity was found in 41% of infants and spontaneous and catheter-associated thrombosis compared to the 12,13 MTHFR1298 heterozygosity in 52% and also were not associated with general population. Factor V Leiden (FVL) is the most 14 increased risk of UC-associated thrombus. The risk of MTHFR double commonly identified risk factor and has been associated with an heterozygosity (db het) was 14%, the risk of a significant or db het was increased incidence of both spontaneous and catheter-associated 12,15–17 17/51 (33%) and the risk of any mutation was 90%. thrombosis. The risk of spontaneous thrombosis increases eightfold in the heterozygous state, and 80-fold in the homozygous Conclusion: Prothrombotic genetic mutations are common in our state.18 Prevalence of heterozygous FVL in the North American Neonatal Intensive Care Unit population but do not appear to increase the Caucasian population is 4 to 6%.19 Prothrombin 20210G>A refers risk of UC-associated thrombosis. to a guanine to adenine point mutation at nucleotide 20210 Journal of Perinatology (2007) 27, 490–495; doi:10.1038/sj.jp.7211786; (PTm) on chromosome 11. This genetic mutation is present in published online 12 July 2007 1 to 2% of the general population but the prevalence is 4 to 20% in selected high-risk groups such as patients with thrombosis.20 Methylene-tetrahydrofolate reductase (MTHFR) 677C>T Correspondence: Dr RM Ryan, Chief, Division of Neonatology, State University of New York at (MTHFR667) and MTHFR1298A>C (MTHFR1298) mutations Buffalo, Women and Children’s Hospital of Buffalo, 219 Bryant Street, Buffalo, NY 14222- cause altered MTHFR activity leading to increased plasma levels of 2006, USA. 16,20–22 E-mail: [email protected] homocysteine, especially in folate-deficient patients. Received 27 October 2006; accepted 18 May 2007; published online 12 July 2007 Hyperhomocystenemia has been associated with an increased risk Prothrombotic mutations and umbilical catheter thrombosis R Turebylu et al 491 of thrombosis.23 The prevalence of MTHFR677 homozygous is 1 to embolism, stroke in young children and any family history of 2% in African Americans and 9.6 to 11% in Caucasians. The heritable thrombophilia. There were no families with a history of heterozygous form is present in 18 to 21.6% of African Americans these problems. Laboratory data included blood counts, blood urea and 41 to 43% of Caucasians.24,25 The prevalence of MTHFR1298 nitrogen, serum creatinine, urine dip stick results, blood cultures, homozygous is 2–4% in African Americans and 9–12.6% in coagulation studies and X-ray/ultrasound results. Caucasians and the heterozygous frequency is 27–30% in African One milliliter of blood was obtained either from the laboratory Americans and 43.6–47% in Caucasians.24,25 The role of (excess sample left over) or from the baby from the UC for genetic heterozygous MTHFR mutations is not clear; however, the mutation studies. Four experienced ultrasonographers performed homocysteine levels also increase in the heterozygous state, duplex Doppler ultrasound scans, using an ATL HDI 5000 suggesting the possibility of increased risk for thrombosis. In ultrasound scanner (ATL Ultrasound, Bothell, WA, USA) to detect addition, the combined heterozygosity of MTHFR 677 and 1298 thrombus on day 3 and days 5 to 7 after catheter placement, and increases homocysteine levels significantly and it has been weekly thereafter, as long as the UC was in situ; a scan was also suggested that this is a possible important risk factor.21,22 performed within 3 days after UC removal. In patients with a UAC, Ultrasound scan has been used successfully to identify large thoracic and abdominal aorta, renal, mesenteric and iliac arteries vessel thrombus.5,26 It is a safe, readily available test that can be were studied. In patients with a UVC, portal vein, hepatic vein and done at the bedside. As umbilical catheters (UCs) have a relatively inferior vena cava (IVC) were studied. All scans were read by the high incidence of associated thrombosis, we hypothesized that same senior radiologist (DM). The size of the thrombus was having one or more of these genetic prothrombotic mutations classified as small (less than 1 cm in length and/or less than 1/4 of (FVL, PTm and/or MTHFR) would increase the risk of the diameter of the aortic lumen), moderate (1 to 2 cm in length UC-associated thrombosis. To our knowledge, this study is the and/or 1/4 to 1/2 of the diameter of the aortic lumen) or large first to examine the prevalence of prothrombotic mutations in an (more than 2 cm in length and/or more than 1/2 of the diameter NICU population requiring UCs, and their relationship with of the aortic lumen). Information from the study ultrasounds was UC-associated thrombosis. not reported routinely to the clinical team caring for the baby. A priori criteria for reporting clinically significant thrombus to the attending neonatologist were devised and included a clot of large Methods size (classified as above) or a clot involving the origin of celiac, This study was a prospective study and was conducted in the NICU renal or mesenteric arteries. at the Women and Children’s Hospital of Buffalo from June 2002 to DNA extraction was performed using a commercially available February 2003. The study was approved by the Institutional Review extraction kit (Gentra Systems Inc., Minneapolis, MN, USA). The Board of the hospital. Written informed consent was obtained from individual loci were amplified using polymerase chain reaction the parents of all enrolled infants. All babies who underwent (PCR) and the genotype was determined using the restriction successful UC insertion as a part of their care were eligible for fragment length polymorphism technique. Quality control inclusion in the study. The decision to place or remove a UC was consisted of three control samples: a known homozygous wild-type, made by the clinical team based on clinical indications. Argyl Neo- heterozygous, homozygous mutant; these samples were included in sert polyurethane catheters 3.5 or 5 F were used to catheterize each PCR run. The genotype for FVL was identified by amplifying a umbilical vessels (Tyco Health Care Group Mansfield, MA, USA). All 287 bp region of the factor V gene and subsequently digested with UAC tip positions were confirmed by X-ray, and were positioned restriction endonuclease MnlI. The amplification of MTHFR between T6 and T9. UVC tips were positioned just above the consisted of 232 bp and digested with HinfI. The prothrombin diaphragm. All UACs had continuous infusion of heparin 1 unit/ genotype was determined by digesting a 345 bp with HindIII. All ml, in half-normal saline, running at 1 ml/h; all UVC solutions digested products were separated by gel electrophoresis on 10% had 0.5 unit/ml of heparin added, and were used to provide routine polyacrylamide.
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