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 -associated

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 , hepatic vein and done at the bedside. As umbilical (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. Each run of participant DNA samples included fluids and parenteral nutrition. three sequenced control DNA samples and a negative control. All All clinical and laboratory data were collected prospectively. The factor V, prothrombin 20210G>A and MTHFR heterozygous and clinical parameters included obstetric factors, presence of homozygous mutant samples were reisolated and reanalyzed to chorioamnionitis, use of antenatal steroids, type of delivery, confirm the genotype. All samples were analyzed according to our gestational age, birth weight, Apgar scores, cord blood gas, type and thrombophilia clinical standard operating procedure manual. length of respiratory support, uses of surfactant, sepsis episodes, necrotizing enterocolitis episodes, intracranial hemorrhage, blood Statistical methods pressure readings, urine output and total length of stay in NICU. At Sample size calculation was based on the assumptions that 22% of the time of consent being obtained, the family was asked patients would be positive for either FVL, PTm or homozygous specifically about any history of deep vein thrombosis, pulmonary MTHFR677, that is significant mutations, (MTHFR1298

Journal of Perinatology Prothrombotic mutations and umbilical catheter thrombosis R Turebylu et al 492 homozygous added later), and that UC-associated thrombus would Table 1 Patient characteristics and umbilical catheter-associated thrombosis occur in 35% of infants. For an a level of 0.05 and a power of 80%, Total Clot present Clot absent we needed to enroll 41 patients (nine cases (significant mutation (n ¼ 53) (n ¼ 16) (n ¼ 37) present) and 32 controls) to be able to detect a threefold increase in UC clot risk in infants with any significant prothrombotic gene Male 25 8 (50%) 17 (46%) mutation. STATA (State College, TX, USA) was used for statistical Female 28 8 (50%) 20 (54%) analysis. Slightly more patients were enrolled (with IRB approval) Mean GA (weeks) 29.9 30.7 29.6 to compensate for problems with blood sample DNA studies since % <32 weeks 35 (66%) 11 (67%) 24 (65%) the blood samples were batched until after the study was Mean birth weight (g) 1520 1638 1496 %<1500 36 (68%) 11 (67%) 25 (68%) completed. Student’s t-test was used to compare continuous data. White 37 (70%) 12 (75%) 25 (68%) w2 test and Fisher’s exact two-tailed probability test were used to Black 16 (30%) 4 (25%) 12 (32%) compare categorical data. Maternal diabetes 1 0 (0%) 1 (2.7%) Preterm labor 38 (72%) 10 (63%) 28 (76%) PROM 11 (21%) 4 (25%) 7 (19%) Results Chorioamnionitis 6 (11%) 1 (6%) 5 (14%) There were 159 neonates who had a UAC or UVC or both placed Antenatal steroids 22 (42%) 5 (31%) 17 (46%) from June 2002 to February 2003, and 70 (based on a convenience Mean 5 min Apgar 7.6 7.8 7.5 sample) were approached for consent. A total of 53 patients were Mean cord pH 7.27 7.24 7.28 enrolled (76% consent rate); 66% were less than 32 weeks Mean cord BE À4.3 À6.1 À3.6 gestation. Of the 53 subjects, 25 had only a UAC, six had only a Hypertension 5 (9%) 0 (0%) 5 (14%) UVC and 22 subjects had both a UAC and UVC, accounting for a Hematuria 7 (13%) 2 (13%) 5 (14%) Oliguriaa 4 (8%) 3 (19%) 1 (3%)b total of 47 UACs and 28 UVCs. A total of 120 Doppler ultrasound Thrombocytopeniac 7 (13%) 3 (19%) 4 (11%) scan examinations were performed on 53 patients. Overall 16 Probable sepsisd 32 (60%) 9 (56%) 23 (62%) (31.3%) had thrombus (either UAC or UVC-related) (Table 1). Proven sepsise 3 (6%) 1 (6%) 2 (5%) Population characteristics such as gestational age, birth weight, use NEC 1 (2%) 1(6%) 0 (0%) of antenatal steroids, Apgar scores, cord pH, hypertension, oliguria, Mean days UAC 4.4 4.8 4.3 necrotizing enterocolitis (NEC), feeding intolerance, sepsis and Mean days UVC 6 6 6 thrombocytopenia did not correlate with the presence of Abbreviations: BE, base excess; PROM, prolonged rupture b-membranes; UAC, umbilical thrombosis. The prevalence of a UAC-associated thrombosis was arterial catheter; UVC, umbilical venous catheter. 6/24 (25%) for babies with a UAC and no UVC vs 5/23 (22%) for aUrine output less than 0.5 ml/kg/h. P-value not significant for any observation. b babies with both a UAC and a UVC. The prevalence of a UVC-related P ¼ 0.06, two-sided Fisher’s exact test. cPlatelet count less than 150 000/ml. thrombosis was 1/6 (17%) for babies with a UVC and no UAC vs dTreated with 7 days of antibiotics at birth although blood culture was negative. 5/22 (23%) for babies with both a UVC and a UAC. eThree infants with UVC had positive blood cultures for coagulase-negative Of the 47 babies with UAC, 11 had arterial thrombus (23.4%). staphylococcus. Of the 11 patients with UAC thrombosis, three were large clots, five were moderate and three were small; none involved the renal Samples for genetic testing were obtained from all 53 infants; arteries. None of the patients with thrombus needed thrombolysis. however, extraction of DNA failed in two patients (neither had Of these 11 babies with aortic thrombus, none had hypertension, thrombus). We included all 53 patients for analyzing the incidence two had hematuria and none had symptoms or signs of acute of and risk factors for thrombosis, but analyzed the 51 patients with vascular compromise. There were five babies with UACs who had complete genetic results for analysis of the relationship of hypertension, but none had thrombus. Of the 28 babies with UVCs, thrombosis with prothrombotic mutations (Figure 1). six had venous thrombus (21.4%). Of the six patients with UVC- The results of genetic testing and relationship with thrombosis associated thrombosis, four had thrombus in the IVC and two had are displayed in Table 2. The prevalence of prothrombotic thrombus in the portal vein. In all of these patients the arterial or mutations in either the heterozygous or homozygous state was venous thrombus was detected only after the removal of the common, being detected in 90% of the population. FVL was catheter. None of the patients needed treatment and all were detected in 8% of the population. None of the babies tested positive asymptomatic. We did not see any significant correlation between for prothrombin 20210G>A. Five patients were homozygous for any of the clinical or laboratory parameters studied and the risk of MTHFR667 (10% of the population). One patient was homozygous thrombosis (Table 1); there was a trend for a higher incidence of for MTHFR1298 (2%). Heterozygous MTHFR667 and MTHFR1298 oliguria in babies with clot present (P-value ¼ 0.06, Fisher’s exact were common, being detected in 41 and 53% of the population two-sided test). respectively. There was no statistically significant increase in the

Journal of Perinatology Prothrombotic mutations and umbilical catheter thrombosis R Turebylu et al 493

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. Figure 1 Restriction enzyme digests to identify mutant factor V. The lanes on Most of our babies with thrombus had either a medium or large the left and right of the gel are the DNA markers increasing by 50 bp. Lanes 2–4 are control samples: (2) normal, (3) homozygous mutant and (4) heterozygous. clot; however most were asymptomatic and treated conservatively. The next five lanes are five patient results: three normal wild-type (wt) and two Although most of the babies had more than one ultrasound scan, heterozygotes (ht) for factor V 1691 G-A. 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 Table 2 Umbilical catheter-associated thrombosis and genetic mutations clinically that clots may be more difficult to visualize while the catheter is still in place. In previous reports, the UVC-associated Total Clot present Clot absent (n ¼ 51) (n ¼ 16) (n ¼ 35) 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 FVL heterozygous 4 (8%) 1 (6%) 3 (9%) incidence of UVC-associated thrombosis in our study was 21.4% MTHFR667 homozygous 5 (10%) 1 (6%) 4 (11%) and is comparable to previous studies. The majority of clots were in MTHFR667 heterozygous 21 (41%) 7 (44%) 14 (40%) the IVC; all babies were asymptomatic and managed conservatively. MTHFR1298 homozygous 1 (2%) 0 (0%) 1 (3%) The prevalence of FVL in our study population was 8%, MTHFR1298 heterozygous 27 (53%) 9 (56%) 18 (51%) comparable to published reports.18,19 The prevalence of the MTHFR double heterozygous 7 (14%) 2 (13%) 5 (14%) MTHFR667 mutation in our study was 41% in the heterozygous Significant mutation: FVL or MTHFR 10 (20%) 2 (13%) 8 (23%) state, and 10% in the homozygous state, again comparable to homozygous 20 Significant or MTHFR db het 17 (33%) 4 (25%) 13 (37%) previously published studies. The prevalence of MTHFR1298 Any mutation 46 (90%) 15 (94%) 31 (89%) mutation in our study was comparable to the wide range of 33 No mutation 5 (10%) 1 (6%) 4 (11%) incidence reported in the United States population. Previous studies have reported an increased prevalence of FVL and MTHFR Abbreviations: db het, double heterozygosity; FVL, factor V Leiden; MTHFR, methylene- tetrahydrofolate reductase. mutations in neonates and children with thrombosis compared to 16,34 None of the patients had prothrombin 20210G>A mutation; P-value not significant for the general population and have also shown an increased risk any comparison between clot present or clot absent. 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. risk of thrombosis in patients carrying any of the mutations being There could be several reasons why our study did not show a tested (Table 2). The same conclusion was found when we correlation between umbilical line-related thrombosis and these analyzed UAC and UVC (Table 3) thrombosis separately, and when common prothrombotic mutations. One is that our sample size was we included only the babies with FVL and homozygous MTHFR small, with only 16 patients having a clot present. Our study was mutations, that is, ‘significant mutations’, and MTHFR double designed to detect a minimum of threefold increase in the risk of heterozygosity (Table 2). 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 Discussion demonstrates that we would be able to detect a 2.5-fold increase in This is the first study to examine the prevalence of prothrombotic risk for these mutations. If the risk is smaller, or specific to site (UA mutations and their effect on UC-associated thrombosis. In this or UV), or true only for one of the mutations, our study may have study, the incidence of UAC-associated thrombosis was 23.4%, missed it. However, there was no suggestion that differences would

Journal of Perinatology Prothrombotic mutations and umbilical catheter thrombosis R Turebylu et al 494

Table 3 Umbilical catheter thrombosis and genetic mutations

Patients with umbilical arterial catheters (n ¼ 45) Patients with umbilical venous catheters (n ¼ 27)

Clot present Clot absent Clot present Clot absent

Total n ¼ 11 n ¼ 34 n ¼ 6 n ¼ 21 FVL heterozygous 0 (0%) 4 (12%) 1 (16%) 1 (5%) MTHFR 667 homozygous 0 (0%) 4 (12%) 1 (16%) 1 (5%) MTHFR 667 heterozygous 6 (55%) 14 (41%) 2 (33%) 7 (33%) MTHFR 1298 homozygous 0 (0%) 1 (3%) 0 (0%) 0 (0%) MTHFR 1298 heterozygous 5 (45%) 18 (53%) 4 (67%) 12 (57%) MTHFR double heterozygous 1 (9%) 6 (18%) 1 (16%) 2 (10%) Significant mutation: FVL or MTHFR homozygous 0 (0%) 9 (26%) 2 (33%) 2 (10%) Significant or MTHFR db het 1 (9%) 15 (44%)a 3 (50%) 4 (19%) Any genetic mutation 10 (91%) 30 (88%) 6 (100%) 17 (81%) No genetic abnormality 1 (9%) 4 (12%) 0 (0%) 4 (19%)

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

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