Fig 2: X-Gal staining of Matr3Gt-ex13 heterozygotes. (A) X-Gal staining of primitive heart (arrow) in E10.5 Matr3 heterozygote, and in CNS (brain, spinal cord), pharyngeal arches and limb bud. (B) X-Gal staining in primitive heart of E8.5 Matr3 heterozygote. Arrow depicts dorsal mesocardium; BC, bulbus cordis; CVC, common ventricular chamber. (C) Negative control wild-type E9.5 embryo with eosin counterstain. (D) X-Gal staining in wall (arrow) of atrial chamber (AC), bulbus cordis (BC) and (E) myocardium and endocardium, and (F) interventricular septum (IVS) of newborn (NB) Matr3GT-ex13 heterozygote heart (arrows).

Fig 3: Clinical features, cardiac phenotype and translocation breakpoint structure in DGAP105. (A) Facial features at 4 years of age included mild hypertelorism, bilateral epicanthal folds, downslanting palpebral fissures, strabismus and a broad nose with a smooth philtrum and thin vermillion border. (B) Ideograms depicting 46,XY,t(1;5)(p36.11;q31.2)dn. Arrows mark locations of AHDC1 and MATR3 breakpoints. (C and D) Echocardiograms at age of 2 days. (C) Ductal view showing distal aortic arch, CoA just distal to the left subclavian artery (LSCA), accompanied by a prominent posterior unfolding (‘posterior shelf’) and PDA. (D) Aortic arch view showing ascending aorta (5.7-mm diameter), hypoplastic aortic arch (4.4-mm diameter) and CoA posterior shelf. (E) Summary of the 1p36.11 and 5q31.2 breakpoints in DGAP105. The 1p36.11 breakpoint disrupts AHDC1 intron 1, whereas the 5q31.2 breakpoint disrupts MATR3 exon 15 in the 3′ UTR. BACs used in FISH analyses are indicated.

Fig 4: Analysis of human MATR3 transcripts and protein expression in control and DGAP105 lymphoblasts. (A) Schematic of the MATR3 exon 13–15 region with the chromosomal translocation breakpoint in patient DGAP105 marked by dotted line. The proximal AAUAAA polyadenylation signal and the distal AAUAAA polyadenylation signal site are shown flanking the breakpoint in the 3′ UTR. TAA denotes the stop codon in exon 15. (B) MATR3 3′ RACE products in human control (Lane 1) and DGAP105 lymphoblast (Lanes 2, 3), and control human fetal heart tissue (Lanes 4, 5). RT ‘+’ or ‘−’ denote inclusion or omission of reverse transcriptase in the cDNA synthesis. The large product (1589 bp, arrow) uses the distal polyadenylation signal and predominates in control human lymphoblasts (Lane 1). In contrast, the short product (963 bp, arrow) predominates in DGAP105 lymphoblasts (Lane 2) and in control human fetal heart (Lane 4) and represents MATR3 transcripts that use the proximal polyadenylation signal. (C) Northern blot analysis of adult human tissues shows MATR3 transcripts of ∼3.5 and ∼2.9 kb. In heart and skeletal muscle, the 2.9-kb transcript predominates and likely corresponds to the 3′ RACE product using the proximal polyadenylation signal. In brain and other tissues, the 3.5-kb transcript predominates and corresponds to the 3′ RACE product using the distal polyadenylation signal. (D) Western blot analysis of protein isolated from DGAP105 and three control lymphoblast lines, showing up-regulation of Matrin 3 in DGAP105 compared with controls. Gapdh was used as loading control. (E) Quantification of Matrin 3 protein expression in D. Bars represent the mean fold expression of four independent experiments ± SEM, corrected for loading, and normalized to Control 2; *P < 0.05 between DGAP105 and mean of the three control lines via paired Student's t test.

Fig 5: Analysis of the Matr3Gt-ex13 gene trap allele. (A) Structure of mouse Matr3 wild-type and Gt-ex13 gene trap mutant alleles. Wild-type mouse Matr3 encodes an 846-amino acid protein. Intron 12 (2749 bp) and exon 13 (223 bp) are shown. Matr3Gt-ex-13 gene trap allele inserts a β-Geo gene trap vector at position 118 bp in exon 13, which will generate Matr3-β-geo fusion transcripts. PCR genotyping primers depict WT-F1 (in exon 13) and WT-R1 (in intron 13) for the wild-type allele, and Mu-F1 (in exon 13) and Mu-R1 (in gene trap vector) for the mutant allele. Primers used in 3′ RACE are summarized on Materials and Methods. (B) E3.5 PCR genotyping shows 394-bp wild-type and 492-bp mutant alleles for wild-type (+/+), heterozygous (+/−) and homozygous (−/−) embryos. (C) Matr3GT-ex13 3′ RACE analysis of mouse E14.5 brain and heart tissues detects a novel Matr3-β-Geo fusion transcript (∼4 kb) in heterozygotes. The long Matr3 3′ RACE product (1647 bp), the only form detected in brain, is reduced in heterozygous brain. Both long and short Matr3 3′ RACE products (1647 and 1025 bp) are reduced in heterozygous heart. (D) Western blot analysis of Matrin 3 protein isolated from wild-type and heterozygous mouse E15.5 brain and heart tissues. Gapdh was used as loading control. (E) Quantification of Matrin 3 protein expression in D. Bars are fold ± SEM expression level from mean of three independent experiments, corrected for loading, and normalized to a value of 1.0 for wild-type heart. The small increase in expression in Matr3Gt-ex13/+ heart is not statistically significant.