ENU induced mutations causing congenital cardiovascular anomalies

Abstract

We used non-invasive high frequency ultrasound to screen N-ethyl-N-nitrosourea mutagenized mouse fetuses for congenital cardiovascular anomalies. We ultrasound scanned 7546 mouse fetuses from 262 mutagenized families, and identified 124 families with cardiovascular defects. Represented were most of the major congenital cardiovascular anomalies seen clinically. The ENU-induced mutations in several families were mapped using polymorphic microsatellite DNA markers. One family with forelimb anomalies and ventricular septal defects, phenotypes similar to Holt-Oram syndrome, and one family with transposition of the great arteries and heart situs anomalies were mapped to different regions of mouse chromosome 4. A third mutation causing persistent truncus arteriosus and craniofacial defects, phenotypes reminiscent of DiGeorge syndrome, was mapped to mouse chromosome 2. We note that mouse chromosomes 4 and 2 do not contain Tbx5 or Tbx1, genes previously linked to Holt-Oram and DiGeorge syndromes, respectively. In two other families, the ENU-induced mutation was identified – Sema3CL605P was associated with persistent truncus arteriosus with interrupted aortic arch, and the Gja1W45Xconnexin43 mutation caused conotruncal malformation and coronary aneurysms. Although our screen was designed as a recessive screen, a number of the mutations showed cardiovascular phenotypes in both heterozygote and homozygote animals. These studies show the efficacy of ENU mutagenesis and high-throughput ultrasound phenotyping in recovering mutations causing a wide spectrum of congenital heart defects. These ENU-induced mutations hold promise in yielding new insights into the genetic basis for human congenital heart disease.

Cardiovascular Assessment of Fetal Mice by In Utero Echocardiography

“…Echocardiographic measurements were previously obtained by serial echocardiography from mouse embryonic day (E) 12.5 to E19.5 (term), with each litter usually scanned three times (Yu et al 2004;Shen et al 2005). The C57BL6/J female mouse usually carried out 5 to 7 fetuses per litter, which is easy to be identified by the ultrasound scanning.…”

“…Ultrasound scanning was conducted as previously described using an Acuson Sequoia C256 ultrasound system with a 15 MHz L8 linear phased array transducer (Acuson Corp., Atlanta, GA, USA) on 2500 fetal mice derived from N-ethyl-N-nitrosurea mutagenized mice (Yu et al 2004;Shen et al 2005). Further information on the mutagenesis and breeding protocols of C57BL6/J mice also can be found at URL: http://pga.jax.org/protocols.html.…”

“…As mice are air breathing mammals with four chamber hearts, they have proven to be an excellent model system for studying human congenital heart disease (Wessels and Sedmera 2003). In fact, most of the major congenital heart anomalies seen clinically can be found in mutant mouse models (Yu et al 2004;Shen et al 2005). Consequently the assessment of fetal mouse cardiac structure and function has become increasingly important for investigating the etiology of human congenital heart disease.…”

“…In this study, we evaluated a large body of echocardiographic data obtained as part of a large scale mouse mutagenesis screen focused on cardiovascular phenotyping (Yu et al 2004;Shen et al 2005). The echocardiographic measurements were acquired by noninvasive in utero ultrasound scanning of mouse fetuses conducted serially using a clinical ultrasound system equipped with a 15 MHz transducer.…”

<em>In utero</em> Measurement of Heart Rate in Mouse by Noninvasive M-mode Echocardiography

“…Mouse is indeed an animal model of choice to study CHD, and most of the human cases can be reproduced in mice 4,5 . Consequently, fetal mouse cardiac phenotyping has become increasingly important to investigate the etiology of human CHD and requires adequate tools.…”