Genetic prothrombotic mutations are common in neonates but ... - Nature

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 1

Division 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

Objective: To evaluate the prevalence of hereditary prothrombotic mutations, and their effect on the incidence and severity of umbilical 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 duplex Doppler ultrasound scan. Genetic testing for factor V Leiden (FVL), prothrombin mutation (PTm) and methylene-tetrahydrofolate reductase (MTHFR) mutations was performed using PCR and restriction fragment length polymorphism assays. Result: Umbilical catheter (UC)-associated thrombosis developed in 16/53 (31%) neonates; 23% of UACs and 22% of UVCs were associated with thrombosis. The prevalence of a significant prothrombotic mutation was present in 10/51 (20%) of infants: FVL (8%), MTHFR667 homozygosity (10%), MTHFR1298 homozygosity (2%) and PTm (0%). There was no increase in the risk of UC-associated thrombus in patients carrying these prothrombotic mutations; our study had the power to detect a 2.5-fold increased risk of thrombosis for any of these significant mutations. In addition, MTHFR667 heterozygosity was found in 41% of infants and MTHFR1298 heterozygosity in 52% and also were not associated with increased risk of UC-associated thrombus. The risk of MTHFR double heterozygosity (db het) was 14%, the risk of a significant or db het was 17/51 (33%) and the risk of any mutation was 90%. Conclusion: Prothrombotic genetic mutations are common in our Neonatal Intensive Care Unit population but do not appear to increase the risk of UC-associated thrombosis. Journal of Perinatology (2007) 27, 490–495; doi:10.1038/sj.jp.7211786; published online 12 July 2007

Correspondence: Dr RM Ryan, Chief, Division of Neonatology, State University of New York at Buffalo, Women and Children’s Hospital of Buffalo, 219 Bryant Street, Buffalo, NY 142222006, USA. E-mail: [email protected] Received 27 October 2006; accepted 18 May 2007; published online 12 July 2007

Keywords: umbilical catheter; thrombosis; duplex Doppler ultrasound; factor V Leiden; methylene-tetrahydrofolate reductase mutation; prothrombin 20210 G>A mutation

Introduction Neonatal umbilical arterial catheter (UAC) and umbilical venous catheter (UVC) usage is routine practice in many neonatal intensive care units (NICUs). UAC usage has been reported in 10–64% of all NICU admissions.1 Thrombosis as a complication of UAC use is common, with an incidence range of 4.5 to 90%.2–5 Complications of aortic thrombosis include gut ischemia, hypertension, decreased renal perfusion, complete aortic occlusion and congestive heart failure.6–9 Thrombosis complicating UVC use is also common, with a reported incidence of 4.1 to 44%.4,10,11 There has been a recent flourish in the identification of genetic abnormalities associated with increased thrombotic risk. Genetic prothrombotic factors are more prevalent in patients with spontaneous and catheter-associated thrombosis compared to the general population.12,13 Factor V Leiden (FVL) is the most commonly identified risk factor14 and has been associated with an increased incidence of both spontaneous and catheter-associated thrombosis.12,15–17 The risk of spontaneous thrombosis increases eightfold in the heterozygous state, and 80-fold in the homozygous state.18 Prevalence of heterozygous FVL in the North American Caucasian population is 4 to 6%.19 Prothrombin 20210G>A refers to a guanine to adenine point mutation at nucleotide 20210 (PTm) on chromosome 11. This genetic mutation is present in 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 (MTHFR667) and MTHFR1298A>C (MTHFR1298) mutations cause altered MTHFR activity leading to increased plasma levels of homocysteine,16,20–22 especially in folate-deficient patients. Hyperhomocystenemia has been associated with an increased risk

Prothrombotic mutations and umbilical catheter thrombosis R Turebylu et al

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of thrombosis.23 The prevalence of MTHFR677 homozygous is 1 to 2% in African Americans and 9.6 to 11% in Caucasians. The heterozygous form is present in 18 to 21.6% of African Americans and 41 to 43% of Caucasians.24,25 The prevalence of MTHFR1298 homozygous is 2–4% in African Americans and 9–12.6% in Caucasians and the heterozygous frequency is 27–30% in African Americans and 43.6–47% in Caucasians.24,25 The role of heterozygous MTHFR mutations is not clear; however, the homocysteine levels also increase in the heterozygous state, suggesting the possibility of increased risk for thrombosis. In addition, the combined heterozygosity of MTHFR 677 and 1298 increases homocysteine levels significantly and it has been suggested that this is a possible important risk factor.21,22 Ultrasound scan has been used successfully to identify large vessel thrombus.5,26 It is a safe, readily available test that can be done at the bedside. As umbilical catheters (UCs) have a relatively high incidence of associated thrombosis, we hypothesized that having one or more of these genetic prothrombotic mutations (FVL, PTm and/or MTHFR) would increase the risk of UC-associated thrombosis. To our knowledge, this study is the first to examine the prevalence of prothrombotic mutations in an NICU population requiring UCs, and their relationship with UC-associated thrombosis. Methods This study was a prospective study and was conducted in the NICU at the Women and Children’s Hospital of Buffalo from June 2002 to February 2003. The study was approved by the Institutional Review Board of the hospital. Written informed consent was obtained from the parents of all enrolled infants. All babies who underwent successful UC insertion as a part of their care were eligible for inclusion in the study. The decision to place or remove a UC was made by the clinical team based on clinical indications. Argyl Neosert polyurethane catheters 3.5 or 5 F were used to catheterize umbilical vessels (Tyco Health Care Group Mansfield, MA, USA). All UAC tip positions were confirmed by X-ray, and were positioned between T6 and T9. UVC tips were positioned just above the diaphragm. All UACs had continuous infusion of heparin 1 unit/ ml, in half-normal saline, running at 1 ml/h; all UVC solutions had 0.5 unit/ml of heparin added, and were used to provide routine fluids and parenteral nutrition. All clinical and laboratory data were collected prospectively. The clinical parameters included obstetric factors, presence of chorioamnionitis, use of antenatal steroids, type of delivery, gestational age, birth weight, Apgar scores, cord blood gas, type and length of respiratory support, uses of surfactant, sepsis episodes, necrotizing enterocolitis episodes, intracranial hemorrhage, blood pressure readings, urine output and total length of stay in NICU. At the time of consent being obtained, the family was asked specifically about any history of deep vein thrombosis, pulmonary

embolism, stroke in young children and any family history of heritable thrombophilia. There were no families with a history of these problems. Laboratory data included blood counts, blood urea nitrogen, serum creatinine, urine dip stick results, blood cultures, coagulation studies and X-ray/ultrasound results. One milliliter of blood was obtained either from the laboratory (excess sample left over) or from the baby from the UC for genetic mutation studies. Four experienced ultrasonographers performed duplex Doppler ultrasound scans, using an ATL HDI 5000 ultrasound scanner (ATL Ultrasound, Bothell, WA, USA) to detect thrombus on day 3 and days 5 to 7 after catheter placement, and weekly thereafter, as long as the UC was in situ; a scan was also performed within 3 days after UC removal. In patients with a UAC, thoracic and abdominal aorta, renal, mesenteric and iliac arteries were studied. In patients with a UVC, portal vein, hepatic vein and inferior vena cava (IVC) were studied. All scans were read by the same senior radiologist (DM). The size of the thrombus was classified as small (less than 1 cm in length and/or less than 1/4 of the diameter of the aortic lumen), moderate (1 to 2 cm in length and/or 1/4 to 1/2 of the diameter of the aortic lumen) or large (more than 2 cm in length and/or more than 1/2 of the diameter of the aortic lumen). Information from the study ultrasounds was 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 size (classified as above) or a clot involving the origin of celiac, renal or mesenteric arteries. DNA extraction was performed using a commercially available extraction kit (Gentra Systems Inc., Minneapolis, MN, USA). The individual loci were amplified using polymerase chain reaction (PCR) and the genotype was determined using the restriction fragment length polymorphism technique. Quality control consisted of three control samples: a known homozygous wild-type, heterozygous, homozygous mutant; these samples were included in each PCR run. The genotype for FVL was identified by amplifying a 287 bp region of the factor V gene and subsequently digested with restriction endonuclease MnlI. The amplification of MTHFR consisted of 232 bp and digested with HinfI. The prothrombin genotype was determined by digesting a 345 bp with HindIII. All digested products were separated by gel electrophoresis on 10% polyacrylamide. Each run of participant DNA samples included three sequenced control DNA samples and a negative control. All factor V, prothrombin 20210G>A and MTHFR heterozygous and homozygous mutant samples were reisolated and reanalyzed to confirm the genotype. All samples were analyzed according to our thrombophilia clinical standard operating procedure manual. Statistical methods Sample size calculation was based on the assumptions that 22% of patients would be positive for either FVL, PTm or homozygous MTHFR677, that is significant mutations, (MTHFR1298 Journal of Perinatology


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homozygous added later), and that UC-associated thrombus would occur in 35% of infants. For an a level of 0.05 and a power of 80%, we needed to enroll 41 patients (nine cases (significant mutation present) and 32 controls) to be able to detect a threefold increase in UC clot risk in infants with any significant prothrombotic gene mutation. STATA (State College, TX, USA) was used for statistical analysis. Slightly more patients were enrolled (with IRB approval) to compensate for problems with blood sample DNA studies since the blood samples were batched until after the study was completed. Student’s t-test was used to compare continuous data. w2 test and Fisher’s exact two-tailed probability test were used to compare categorical data. Results There were 159 neonates who had a UAC or UVC or both placed from June 2002 to February 2003, and 70 (based on a convenience sample) were approached for consent. A total of 53 patients were enrolled (76% consent rate); 66% were less than 32 weeks gestation. Of the 53 subjects, 25 had only a UAC, six had only a UVC and 22 subjects had both a UAC and UVC, accounting for a total of 47 UACs and 28 UVCs. A total of 120 Doppler ultrasound scan examinations were performed on 53 patients. Overall 16 (31.3%) had thrombus (either UAC or UVC-related) (Table 1). Population characteristics such as gestational age, birth weight, use of antenatal steroids, Apgar scores, cord pH, hypertension, oliguria, necrotizing enterocolitis (NEC), feeding intolerance, sepsis and thrombocytopenia did not correlate with the presence of thrombosis. The prevalence of a UAC-associated thrombosis was 6/24 (25%) for babies with a UAC and no UVC vs 5/23 (22%) for babies with both a UAC and a UVC. The prevalence of a UVC-related thrombosis was 1/6 (17%) for babies with a UVC and no UAC vs 5/22 (23%) for babies with both a UVC and a UAC. Of the 47 babies with UAC, 11 had arterial thrombus (23.4%). Of the 11 patients with UAC thrombosis, three were large clots, five were moderate and three were small; none involved the renal arteries. None of the patients with thrombus needed thrombolysis. Of these 11 babies with aortic thrombus, none had hypertension, two had hematuria and none had symptoms or signs of acute vascular compromise. There were five babies with UACs who had hypertension, but none had thrombus. Of the 28 babies with UVCs, six had venous thrombus (21.4%). Of the six patients with UVCassociated thrombosis, four had thrombus in the IVC and two had thrombus in the portal vein. In all of these patients the arterial or venous thrombus was detected only after the removal of the catheter. None of the patients needed treatment and all were asymptomatic. We did not see any significant correlation between any of the clinical or laboratory parameters studied and the risk of thrombosis (Table 1); there was a trend for a higher incidence of oliguria in babies with clot present (P-value ¼ 0.06, Fisher’s exact two-sided test). Journal of Perinatology

Table 1 Patient characteristics and umbilical catheter-associated thrombosis

Male Female Mean GA (weeks) % A mutation; P-value not significant for any comparison between clot present or clot absent.

risk of thrombosis in patients carrying any of the mutations being tested (Table 2). The same conclusion was found when we analyzed UAC and UVC (Table 3) thrombosis separately, and when we included only the babies with FVL and homozygous MTHFR mutations, that is, ‘significant mutations’, and MTHFR double heterozygosity (Table 2). Discussion This is the first study to examine the prevalence of prothrombotic mutations and their effect on UC-associated thrombosis. In this study, the incidence of UAC-associated thrombosis was 23.4%,

comparable to previous studies.2–6,27 UACs have been in use for more than 40 years, with the reported incidence of thrombosis being almost constant, in spite of advances in the types of material used to make UACs.28 The reported risk factors for thrombosis include the presence of an intravascular catheter,29 duration of catheterization,4 infusion of calcium containing fluids5 and dehydration, sepsis, polycythemia or congenital heart disease.30 Horgan et al.31 reported that continuous infusion of heparin, which is a standard practice worldwide, will not decrease the risk of thrombosis, but will prolong the patency of the catheter. In our study population, we did not see any specific risk factors for thrombosis. The mean duration of UAC use was short at 4.4 days. We do not use calcium-containing fluids in UACs in our NICU. Most of our babies with thrombus had either a medium or large clot; however most were asymptomatic and treated conservatively. Although most of the babies had more than one ultrasound scan, only the last scan performed, after the removal of the catheter, revealed the arterial or venous clot. The scans were performed by very experienced ultrasonographers. It may be important to note clinically that clots may be more difficult to visualize while the catheter is still in place. In previous reports, the UVC-associated thrombosis incidence depends on the type of the study and the timing of the study in relation to catheter removal.4,10,27,32 The incidence of UVC-associated thrombosis in our study was 21.4% and is comparable to previous studies. The majority of clots were in the IVC; all babies were asymptomatic and managed conservatively. The prevalence of FVL in our study population was 8%, comparable to published reports.18,19 The prevalence of the MTHFR667 mutation in our study was 41% in the heterozygous state, and 10% in the homozygous state, again comparable to previously published studies.20 The prevalence of MTHFR1298 mutation in our study was comparable to the wide range of incidence reported in the United States population.33 Previous studies have reported an increased prevalence of FVL and MTHFR mutations in neonates and children with thrombosis compared to the general population16,34 and have also shown an increased risk of catheter-related thrombosis in patients with FVL;12,13 however, we found no difference in the prevalence of MTHFR mutations and FVL mutation in infants with and without thrombosis. There could be several reasons why our study did not show a correlation between umbilical line-related thrombosis and these common prothrombotic mutations. One is that our sample size was small, with only 16 patients having a clot present. Our study was designed to detect a minimum of threefold increase in the risk of genetic mutation-associated thrombosis for one of the mutations known to increase the risk of thrombosis (FVL, PTm, MTHFR homozygous); with the actual results obtained, a power calculation demonstrates that we would be able to detect a 2.5-fold increase in risk for these mutations. If the risk is smaller, or specific to site (UA or UV), or true only for one of the mutations, our study may have missed it. However, there was no suggestion that differences would Journal of Perinatology


494 Table 3 Umbilical catheter thrombosis and genetic mutations Patients with umbilical arterial catheters (n ¼ 45)

Total FVL heterozygous MTHFR 667 homozygous MTHFR 667 heterozygous MTHFR 1298 homozygous MTHFR 1298 heterozygous MTHFR double heterozygous Significant mutation: FVL or MTHFR homozygous Significant or MTHFR db het Any genetic mutation No genetic abnormality

Clot present

Clot absent

n ¼ 11 0 (0%) 0 (0%) 6 (55%) 0 (0%) 5 (45%) 1 (9%) 0 (0%) 1 (9%) 10 (91%) 1 (9%)

n ¼ 34 4 (12%) 4 (12%) 14 (41%) 1 (3%) 18 (53%) 6 (18%) 9 (26%) 15 (44%)a 30 (88%) 4 (12%)

Patients with umbilical venous catheters (n ¼ 27) Clot present

Clot absent

n¼6 (16%) (16%) (33%) (0%) (67%) (16%) (33%) (50%) (100%) (0%)

n ¼ 21 1 (5%) 1 (5%) 7 (33%) 0 (0%) 12 (57%) 2 (10%) 2 (10%) 4 (19%) 17 (81%) 4 (19%)

1 1 2 0 4 1 2 3 6 0

Abbreviations: db het, double heterozygosity; FVL, factor V Leiden; MTHFR, methylene-tetrahydrofolate reductase. P-value was not significant for any observation. a P ¼ 0.067, two-sided Fisher’s exact test, 0.034 one-sided.

be found with a larger number of patients. We also did not test for all of the prothrombotic risk factors such as lipoprotein-a, protein C, protein S and antithrombin III abnormalities. Hyytiainen et al.35 have shown that the procoagulant effect of FVL may be balanced against decreased levels of Factor V in newborns compared to adults, and this may explain the lack of an increased risk of thrombosis in babies positive for FVL. Finally, it may be that newborns have additional risk factors for thrombosis that overshadow the specific MTHFR and FVL abnormalities studied. In conclusion, both UAC and UVC-related thrombosis were common (23 and 21% respectively) and were essentially asymptomatic and clinically unsuspected. Of note, in this study all thromboses were identified after catheter removal. The prevalence of prothrombotic genetic mutations is common in our NICU population but does not appear to increase the risk of UC-associated thrombosis.

Acknowledgments We thank sonographers Yvonne B Machajewski, Patricia Rusin-Sebo, Linda Malobecki and Mary Baldo for their help with sonograms. We thank Frederick C Morin III MD, Interim Dean, School of Medicine and Biomedical Sciences, SUNY at Buffalo for his interest in and support of this project.

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