Background: The cardiac myosin binding protein C gene is mutated in Maine Coon (MC) cats with familial .... cats and Maine Coon cross cats with the MYBPC3 mutation and left ..... Marian AJ, Salek L, Lutucuta S. Molecular genetics and.
J Vet Intern Med 2007;21:232–237
Tissue Doppler Imaging in Maine Coon Cats with a Mutation of Myosin Binding Protein C with or without Hypertrophy Kristin A. MacDonald, Mark D. Kittleson, Philip H. Kass, and Kathryn M. Meurs Background: The cardiac myosin binding protein C gene is mutated in Maine Coon (MC) cats with familial hypertrophic cardiomyopathy. Hypotheses: Early diastolic mitral annular velocity is incrementally reduced from normal cats to MC cats with only an abnormal genotype to MC cats with abnormal genotype and hypertrophy. Animals: Group 1 consisted of 6 normal domestic shorthair cats, group 2 of 6 MC cats with abnormal genotype but no hypertrophy, and group 3 of 15 MC cats with hypertrophy and abnormal genotype. Methods: The genotype and echocardiographic phenotype of cats were determined, and the cats were divided into the 3 groups. Tissue Doppler imaging (TDI) of the lateral mitral annulus from the left apical 4-chamber view was performed. Five nonconsecutive measurements of early diastolic mitral annular velocity (EM) or summated early and late diastolic velocity (EAsum) and heart rate were averaged. Results: There was an ordered reduction in Em-EAsum as group number increased (group 1, range 9.7–14.7 cm/s; group 2, range 7.5–13.2 cm/s; group 3, range 4.5–14.1 cm/s; P 5 .001). Using the lower prediction limit for normal Em-EAsum, the proportion of cats with normal Em-EAsum decreased as the group number increased (P 5 .001). However, Em-EAsum was reduced in only 3 of 6 cats in group 2. Conclusion: The incremental reduction of Em-EAsum as group severity increased indicates that diastolic dysfunction is an early abnormality that occurs before hypertrophy development. TDI measurement of Em or EAsum of the lateral mitral annulus is an insensitive screening test for identification of phenotypically normal, genotypically affected cats. Key words: Diastolic function; Genotype; Phenotype; Hypertrophic cardiomyopathy.
ypertrophic cardiomyopathy (HCM) is the most common heart disease of cats and is inherited as an autosomal dominant trait in a family of Maine Coon cats.1 The causative mutation of HCM in this family of Maine Coon cats is a missense mutation in the sarcomeric protein myosin binding protein C gene (MYBPC3), that results in a change from the conserved amino acid alanine to proline, thus altering protein conformation.2 Myosin binding protein C is located at the transverse band within the A band of the sarcomere and attaches to titin and B-myosin heavy chain. It is believed to have both structural and regulatory roles.3 Mutations in MYBPC3 are the most common cause of familial HCM in people, and occur in 14–26% of familial cases.4 Incomplete penetrance is common with mutations in this gene, often making echocardiographic diagnosis of HCM difficult in heterozygous individuals.3 Maine Coon cats with HCM develop concentric hypertrophy, myofiber disarray, interstitial and replacement fibrosis, and possibly left atrial enlargement.1 Systolic anterior motion of the mitral valve is common. Cats with HCM have impaired relaxation and diastolic dysfunction evident on traditional echocardiography and tissue Doppler imaging (TDI) echocardiography.5,6
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From the Departments of Medicine and Epidemiology (MacDonald, Kittleson) and Population Health and Reproduction (Kass), School of Veterinary Medicine, University of California, Davis, Davis, CA; and the Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, WA (Meurs). Reprint requests: Kristin MacDonald, Animal Care Center of Sonoma, 6470 Redwood Dr., Rohnert Park, CA 94928; e-mail: kamacdonald@ucdavis.edu. Submitted November 30, 2005; Revised May 4, 2006, September 27, 2006; Accepted November 7, 2006. Copyright E 2007 by the American College of Veterinary Internal Medicine 0891-6640/07/2102-0005/$3.00/0
TDI echocardiography has emerged as one of the most sensitive and specific methods for noninvasive assessment of diastolic function and is relatively unaffected by loading conditions.7 Diastolic function is most commonly assessed by measuring the early diastolic velocity of the mitral annulus (Em).8–11 Em is reduced in cats with HCM when compared to normal cats.6,10 Em also correlates with invasive measurements of diastolic function in cats.12 Potential pathophysiologic consequences of severe HCM and diastolic dysfunction include development of congestive heart failure and systemic thromboembolism. Population screening and early recognition of HCM has become important in human medicine and in certain breeds of cats that appear predisposed to HCM. Until now, HCM diagnosis in cats was dependent on identification of left ventricular concentric hypertrophy by echocardiography in the absence of other diseases that cause hypertrophy. The use of a genetic screening test for the mutation of MYBPC3 is useful to identify genotypically affected Maine Coon cats within this colony.2 However, there are no genotypic screening tests for familial HCM in purebred cats other than Maine Coon cats. Because there are more than 200 mutations of 10 sarcomeric genes in people, it is likely that each breed will have a different mutation and identifying them will be a long and laborious process.13 Therefore, identification of left ventricular concentric hypertrophy by echocardiography will remain the fundamental basis of diagnosis of HCM in cats for some time. However, TDI echocardiography might be a useful earlier screening modality to identify diastolic and systolic abnormalities in familial HCM before development of concentric hypertrophy because it is abnormal in humans and other animal models before the development of left ventricular wall thickening.14,15 The hypothesis of the study was that cats with the identified mutation of MYBPC3 without phenotypic
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Fig 1. Pulsed-wave tissue Doppler imaging (TDI) in normal cats and cats with hypertrophic cardiomyopathy. Pulsed-wave TDI was performed at the lateral mitral annulus using a left apical 4-chamber view with the gate placed perpendicular to the motion of the heart (A). TDI of a normal cat in group 1 shows fusion of the early and late diastolic velocities into a single EAsum wave, and there was a rapid heart rate of 220 bpm (B). TDI of a Maine Coon cat with severe hypertrophic cardiomyopathy without heart failure in group 3, showing reduced Em velocity and E : A reversal, which indicate diastolic dysfunction, and there was a slow heart rate of 104 bpm (C). EA, summated early and late diastolic velocity wave; S, systolic wave; Em, early diastolic mitral annular velocity, A, late diastolic mitral annular velocity.
evidence of hypertrophy have diastolic dysfunction when assessed by TDI echocardiography. Specific aims were to determine if there was an ordered decrease in diastolic function from normal cats to cats with the mutation but no hypertrophy to cats with the mutation and hypertrophy, and to determine if measurement of early diastolic mitral annular velocity was a useful means of screening cats without hypertrophy for the mutation.
Materials and Methods Study Population This study consisted of normal domestic shorthair (DSH) cats (group 1), Maine Coon cross cats with the MYBPC3 mutation in the absence of left ventricular hypertrophy (group 2), and Maine Coon cats and Maine Coon cross cats with the MYBPC3 mutation and left ventricular hypertrophy (group 3). Group 1 cats were normal DSH cats residing within another research colony and were unrelated to the Maine Coon and Maine Coon cross cats. Unrelated DSH cats were chosen for group 1 because Maine coon cats and Maine coon cross cats residing in the HCM colony could not be declared as normal based on a normal genotype for MBYPC3. There is another mutation within the colony that causes HCM, and the mutation has yet to be defined. Therefore, it was necessary to use unrelated DSH cats in the control group. Maine Coon cats and Maine Coon cross cats in group 2 and group 3 live in a research colony of cats with familial hypertrophic cardiomyopathy. This numbering was employed to indicate the ordering of relative severity of clinical disease among groups in this study population.
Mutational Analysis Two milliliters of blood was collected from Maine Coon cats residing in a familial HCM research colony. DNA was extracted from peripheral lymphocytes from all cats as previously described.16 An oligonucleotide was designed for amplification of exon 3 of the MYBPC3 gene in cats, using known human sequences (GenBank U91629) and Primer3 software.17 The exon was amplified at 95uC (5 minutes) followed by 40 cycles of 94uC (20 seconds), 57uC (20 seconds), and 74uC (39 seconds). The polymerase chain reaction product was run on an agarose gel, cut from the gel, and purified with the QiaQuick kit.a Restriction enzyme digests were performed to confirm the identification of the mutation by running 10 mL of the sample combined with 3-mL HaeIII buffer, 5.5-mL water, and 1.5-mL HaeIII, an enzyme that
cuts specifically at a GGCC region, and incubated at 37uC for 2 hours. The affected cats have a mutation that replaces the second G and prevents the enzyme from cutting an appropriate sized fragment compared to normal cats. Sixteen microliters of the sample was run on a polyacrylamide gel for 45 minutes to 1hour at 250 V. The gel was placed into an ethidium bromide solution for 5 minutes, rinsed in distilled water for 5 minutes, and viewed under ultraviolet light to evaluate the fragment sizes. Unaffected cats were identified by the presence of 50 and 55 base pair sized fragments, affected cats were identified by the presence of 50 and 75 base pair fragments. Samples from affected cats were subsequently genotyped by sequencing on an ABI377 sequencer,b and the sequence was evaluated to determine if they were heterozygous (G/CCC) or homozygous (CCC) for the mutation.2
Echocardiography Standard echocardiography was performed on all affected cats while sedated with 0.1 mg/kg acepromazine and 0.1 mg/kg hydromorphone SC.c A left ventricular free wall end diastolic thickness (LVFWd) or an interventricular septal end diastolic thickness (IVSd) .6 mm was defined as abnormal. Left atrial and aortic diameters were measured by 2-dimensional echocardiography of the right parasternal short-axis basilar view, and left atrial dilation was defined as the ratio of left atrium to aortic diameter (LA:Ao) $1.5. Systolic blood pressure was measured in all cats with concentric hypertrophy and had to be within the normal range of ,160 mmHg for a cat to be included in the study.d The metatarsal region of one hind limb of each cat was shaved, and a 3-cm cuff was placed above the tarsus. Serial blood pressure measurements were made for 5 minutes, and the lowest consistent value obtained in 3 measurements was chosen.
Tissue Doppler Imaging Pulsed-wave TDI of the lateral mitral annulus from the leftapical 4-chamber view was performed using a 12-MHz probe, with the pulsed-wave Doppler gate placed perpendicular to myocardial motion (Fig 1).c Specific TDI settings included: Nyquist limit 10– 15 cm/s; sweep speed 100 cm/s; gate width 0.11 cm; and filter 50 Hz. Heart rate (HR) was measured by an electrocardiogram. Five nonconsecutive measurements of Em or summated early and late diastolic velocity (EAsum) of the lateral mitral annulus were recorded and averaged (Fig 1). The HRs of the 5 nonconsecutive measurements were also averaged. Early and late diastolic mitral annular velocity waves fuse when there are high HRs, preventing
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Table 1. Echocardiographic measurements of normal domestic shorthair cats (group 1), Maine Coon cats and Maine Coon cross cats with myosin binding protein C mutation and no left ventricular hypertrophy (group 2), or with hypertrophy (group 3).
Echocardiographic Measurement
Group 1 (n 5 6) 2Genotype 2Phenotype Median (Range)
LVFWd (mm) IVSd (mm) Em (cm/s) HR (beats/min)
4.8 5.7 11.6 204
(4–5.3) (4–6) (9.7–14.7) (143–260)
Group 2 (n 5 6) +Genotype 2Phenotype Median (Range) 4.7 4.5 8.4 172
(4–5.1) (3.8–5.3) (7.5–13.2) (157–244)
Group 3 (n 5 15) +Genotype +Phenotype Median (Range) 6.6 6.3 7.7 175
(4.7–7.8) (4.1–7.6) (4.5–14.1) (101–256)
LVFWd, left ventricular free wall end diastolic thickness; IVSd, interventricular septal end diastolic thickness; Em, early diastolic mitral annular velocity; HR, heart rate. the measurement of individual Em waves in many cats (Fig 1B). The tracings that were chosen had the highest velocities and minimal artifact. The operator (KM) obtaining and reading the echocardiogram and TDI was blinded to the group number of the cat, with the exception of group 1 cats. Normal reference values of Em-EAsum were obtained by another investigator from 20 normal, awake DSH cats.e,6 Sixty-four measurements of Em-EAsum were made at HRs ranging from 115– 242 bpm.6 Because Em-EAsum is positively correlated with HR, 95% prediction intervals were constructed to determine the upper and lower limits of normal Em-EAsum depending on the HR, using the following formulas: vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u u Þ2 1 ðX { X SIND ~ SY:Xu P 2 t1 z n z P ð Xi Þ Xi2 { n
Prediction interval 5 Yc 6 tSIND Where_ Y is predicted individual value of Em-EAsum; X, heart rate; X, mean heart rate; n, sample number; SIND, square root of variance of Y; and t, the t multiple determined for n-2 degrees of freedom.
Statistical Analysis The Jonckheere-Terpstra test was used to assess the presence of a continuous Em-EAsum response according to ordinal group, ie, that Em-EAsum decreases as group number (severity) increases. The Kruskal-Wallis test for singly ordered contingency table data was used to compare ordinal groups (group 1, then group 2, then group 3) to a dichotomous grouping of EM-EAsum (normal or reduced). Em-EAsum was defined as normal or reduced by using 95% prediction intervals of Em-EAsum depending on HR. Kruskal-Wallis 1-way analysis of variance was used to assess whether HR and age were different among the 3 groups of cats. Sensitivity and specificity of Em-EAsum for detecting genotypically affected cats was calculated. A level of significance was defined as P , .05. Em-Easum and HR was obtained 2 separate days within 10 days by the same operator in 10 Maine Coon and Maine Coon cross cats. Time-intraobserver differences were statistically assessed by a paired t-test.f
Results Study Population There were 6 normal DSH cats (Group 1) that were unrelated to the Maine Coon cats and Maine Coon cross
cats with the MYBPC3 mutation. Median values of wall thickness of group 1 cats were LVFWd 4.8 mm (range 4–5.3 mm) and IVSd 5.7 mm (range 4–6 mm) (Table 1). All cats in group 1 had normal left atrial size. There were 6 Maine Coon cross cats (3 male and 3 female) with a mutation of MYBPC3 but no phenotypic evidence of left ventricular hypertrophy (group 2). All cats in group 2 were heterozygous for the MYBPC3 mutation. No cats in group 2 had left ventricular hypertrophy, papillary hypertrophy, or left atrial enlargement (median LVFWd 4.7 mm, range 4–5.1 mm; median IVSd 4.5 mm, range 3.8–5.3 mm) (Table 1). Last, there were 15 Maine Coon cats and Maine Coon cross cats (7 male and 8 female) with MYBPC3 mutation and phenotypic evidence of left ventricular hypertrophy (group 3). One cat in group 3 was homozygous for the MYBPC3 mutation, and the remaining 14 cats were heterozygous for the mutation. All cats in group 3 had mild to severe concentric left ventricular hypertrophy (LVFWd median 6.6 mm, range 4.7–7.8 mm; IVSd median 6.3 mm, range 4.1–7.6 mm), and 3 cats (20%) had mild to severe left atrial enlargement (LA/Ao 1.6, 1.8, and 2.0, respectively) (Table 1). Median ages and age ranges of cats in group 1, group 2, and group 3 were 3.5 years (3–4 years), 5.5 years (2.8–6.1 years), and 8.4 years (2.4–12.9 years), respectively. There was no difference in ages among the 3 groups (P 5 .35).
Tissue Doppler Imaging Em-EAsum decreased as group number (ie, severity of disease) increased, meaning that there was an ordered response of Em-EAsum depending on the group number (group 1, median 11.6 cm/s, range 9.7–14.7 cm/s; group 2, median 8.4 cm/s, range 7.5–13.2 cm/s; group 3 median 7.7; range 4.5–14.1; P 5 .001) (Table 1, Fig 2). Using the 95% prediction intervals for the lower limit of normal Em depending on HR, no normal cats, 3 cats (50%) in group 2, and 12 cats (80%) in group 3 had abnormally low Em-EAsum relative to HR (Fig 3). Using the Kruskal-Wallis test for singly ordered contingency table data, there was an ordered difference in the number of cats with normal Em-EAsum depending on group number, such that the number of cats with normal Em-EAsum decreased as the group number increased (P 5 .001). Using the Kruskal-Wallis test, HR
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(80%) and specific (100%) for detection of the affected genotype in cats in group 3. There was no time-intraobserver difference in EmEAsum (P 5 .9) or HR (P 5 .07) in 10 Maine Coon and Maine Coon cross cats that were examined twice within a 10-day period by the same (blinded) operator.
Discussion
Fig 2. Box and whisker plots of early mitral annular diastolic velocity in normal domestic shorthair cats (group 1), Maine Coon cats and Maine Coon cross cats with myosin binding protein C mutation without left ventricular hypertrophy (group 2), or with hypertrophy (group 3). As group number (X axis) increased, early mitral annular velocity (Em) decreased (P 5 .001; Group 1, median 11.6 cm/s, range 9.7–14.7 cm/s; Group 2, median 8.4 cm/s, range 7.5–13.2 cm/s; Group 3, median 7.7 cm/s, range 4.5–14.1 cm/s).
was not different among the 3 groups (P 5 .49; group 1, median 204 bpm, range 143–260 bpm; group 2, median 179 bpm, range 158–244 bpm; group 3, median 175 bpm, range 101–250 bpm). TDI measurement of Em-EAsum of the lateral mitral annulus was insensitive (50%) but specific (100%) for detection of the affected genotype in group 2 cats. TDI measurement of Em-EAsum was both highly sensitive
Fig 3. Tissue Doppler imaging echocardiography measurement of early diastolic myocardial velocity at the lateral mitral annulus in normal domestic shorthair cats (group 1), Maine Coon cats and Maine Coon cross cats with a myosin binding protein C mutation without left ventricular hypertrophy (group 2) or with hypertrophy (group 3). (under fig): As group number increased, early diastolic mitral annular velocity (Em) decreased (P 5 .001). Maine Coon cats and Maine Coon cross cats with a mutation in the myosin binding protein C gene without hypertrophy (group 2) had an intermediate reduction in Em compared to the normal domestic shorthair cats (group 1), and Maine Coon cats and Maine Coon cross with the MYBPC3 mutation and hypertrophy (group 3) had the lowest early diastolic mitral annular velocity (Em). Heart rate was not significantly different among the 3 groups (P 5 .49). The solid line represents the 95% prediction interval for lower limit of normal Em depending on heart rate. Based on the lower prediction limit for normal Em, the number of cats with normal Em decreased as the group number increased (P 5 .001).
This study found that there was an ordered decrease in Em-EAsum that corresponded to the increase in group number, ie, as the group number increased from normal to genotype-positive phenotype-negative to genotype and phenotype positive, Em-EAsum decreased. As has been shown previously, Em-EAsum was also reduced in Maine Coon cats and Maine Coon cross cats with the MYBPC3 mutation with concentric hypertrophy.18 This study demonstrates that diastolic dysfunction can be an early component of the pathophysiology of HCM rather than merely a consequence of left ventricular hypertrophy and fibrosis. However, on an individual basis, Em-EAsum of the lateral mitral annulus was reduced in only 50% (3/6) of the cats with the mutation of MYBPC3 without hypertrophy and is therefore not a sensitive enough screening test (sensitivity 50%) for detection of genotypically affected cats with no hypertrophy. TDI measurement of Em-EAsum is a very sensitive test (80%) for detection of genotypically affected cats with hypertrophy. These findings are consistent with a previous report that identified impaired systolic and diastolic function using TDI in a mutant B-myosin heavy chain transgenic rabbit model of HCM before development of concentric hypertrophy.15 There are also several small studies evaluating the use of TDI measurement of diastolic function as a screening test for identification of genotypically affected people with familial HCM in the absence of hypertrophy.19,20 In one study of 13 people with a mutation for familial HCM but no hypertrophy, 30 people with a mutation and concentric hypertrophy, and 30 age-matched controls, Em was 100% sensitive and 90% specific for the diagnosis of people with only the abnormal genotype.19 Another study using TDI to predict genotype (B-myosin heavy chain mutation) in people with preclinical HCM found a substantial overlap of EM velocities between genotypically affected people without hypertrophy (n 5 18) and normal people (n 5 18), with a sensitivity of 75% and a specificity of 86% for detection of the affected genotype.20 Another study revealed that TDI is predictive of HCM development in genotypically affected people, again without evidence of hypertrophy.14 People with a lower baseline Ea velocity had a greater increase in left ventricular mass across 2 years (R 5 20.86).14 Further follow-up of the genotypically affected cats without hypertrophy in the current study would be useful to identify whether the same relationship exists between baseline Ea and subsequent development of hypertrophy in cats. One hypothesis in familial HCM is that the initial phenotype is a functional sarcomeric defect and there
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are intermediary pathways that connect the initial defect to the final phenotype of left ventricular hypertrophy, myocardial fibrosis, and myofiber disarray.3 Gene transfer studies in adult rat ventricular cardiomyocytes expressing HCM-associated mutant troponin T protein have demonstrated myocyte dysfunction before development of myofibrillar disarray.21,22 Impaired cardiomyocyte mechanical function leads to increased myocyte stress and activation of stress-responsive intracellular signaling kinases, calcium-sensitive signaling molecules, and trophic factors.3 Transcriptional machinery of the myocyte is activated, which leads to myocyte hypertrophy, collagen synthesis, and myocyte disarray. Left ventricular hypertrophy is a compensatory process occurring later in the disease in familial HCM models. Dysfunctional myosin binding protein C (cMyBP-C) protein may negatively impact the structure and function of the sarcomere.23 The axial alignment of cMyBP-C along the B2MHC backbone and the interaction of cMyBP-C with titin are necessary for ordered, stabilized arrangement of the sarcomere. Consequently, the absence of cMyBP-C in transgenic cMyBP-C knockout mice resulted in malalignment of the sarcomeric striations.23 cMyBP-C also interacts with the beta-myosin heavy chain (b-MHC) head and acts as a braking mechanism between the interaction of actin and b2MHC. When cMyBP-C is phosphorylated, it undergoes a conformational change in the C0–C1 linker region that releases the myosin head to be in a favorable position to bind with actin.24 The mutation in MYBPC3 in Maine Coon cats was localized to the C0 and C0–C1 linker region involved with binding to myosin, actin, or both.2 In an experimental model of interrupted cMyBPC and myosin interaction in ventricular myocytes, there was increased calcium sensitivity, force of contraction, and time to half-relaxation.25 Similarly, in a knock-in mouse familial HCM model missing the linker between motifs C0–C1, there was an increased calcium sensitivity to force production.26 These experimental findings may help identify possible pathophysiologic mechanisms of familial HCM in Maine Coon cats with mutation of MYBPC3. There were several limitations of this study. TDI measurement only included the lateral mitral annulus early diastolic velocity or summated early and late diastolic velocities, which is an index of global diastolic function of the longitudinal muscle fibers, and did not include measurement of other regions of the left ventricle such as the interventricular septum or the left ventricular free wall. Em waves could not be measured in all cats because of the summation of EA waves at higher HRs, and therefore it was necessary to compare Em or EAsum among groups. However, the normal prediction intervals calculated for Em-EAsum depending on HR reflected EAsum waves that occurred in normal cats at high HRs. Because EA summation occurred in many cats, measurements of A waves or E : A ratio could not be determined. Pulsed-wave TDI was used, which did not allow calculation of myocardial velocity gradients or strain rate. It is possible that other indices of diastolic function may have been impaired in
Maine Coon cats that were genotypically affected but phenotypically normal. Because of the limited number of normal cats and the need to measure Em-EAsum across a wide range of HRs, it was necessary to measure Em-EAsum several times in some cats. Repeated measures of Em-EAsum within individual normal cats may falsely narrow prediction intervals if there are cat-specific effects of HR on EM-EAsum. The number of normal DSH cats in group 1 was small. Systolic blood pressure was not measured in the group 1 and group 2 cats that did not have evidence of concentric hypertrophy. These cats were young, overtly healthy, and free of any clinical signs. In conclusion, this study found that Maine Coon cats and Maine Coon cross cats with a MYBPC3 mutation have incrementally reduced Em depending on the absence or presence of hypertrophy as compared with normal DSH cats. Cats that were genotypically affected with no hypertrophy had intermediate Em values as a group compared to normal DSH cats and cats that were genotypically affected and had hypertrophy, suggesting that the pathophysiology of the disease in Maine Coon cats may be similar to that seen in humans and genetic models of HCM. However, on an individual level, TDI is an insensitive screening test to identify genotypically affected cats before the presence of hypertrophy.
Footnotes a
QiaQuick, Qiagen Inc, Spoorstraat 50, KJ Venlo 5911, Netherlands b ABI377 sequencer, Applied Biosystems, Foster City, CA c HP Sonos 5500, Philips Medical Systems, Andover, MA d Parks Medical Electronics, Inc, Aloha, OR e Acuson 128XP/10, upgraded with Acoustic Response Technology, Acuson DTI software, and Regional Expansion Selection, Acuson Corps, Mountain View, CA f StatXact, Version 6, Cytel Software Corporation, Cambridge, MA
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