Identification and molecular characterization of de novo translocation t(8;14)(q22.3;q13) associated with a vascular and tissue overgrowth syndrome

Abstract

Klippel-Trenaunay syndrome (KTS) is a disorder primarily characterized by capillary-venous vascular malformations associated with altered limb bulk and/or length. We report the identification of a balanced translocation involving chromosomes 8q22.3 and 14q13 in a patient with a vascular and tissue overgrowth syndrome consistent with KTS. We demonstrated that translocation t(8;14)(q22.3;q13) arose de novo. These data suggest that a pathogenic gene for a vascular and tissue overgrowth syndrome (KTS) may be located at chromosome 8q22.3 or 14q13. Fluorescence in situ hybridization (FISH) analysis was used to define the breakpoint on chromosome 8q22.3 to a <5-cM interval flanked by markers AFMA082TG9 and GATA25E10, and the 14q13 breakpoint within a 1-cM region between STSs WI-6583 and D14S989. This study provides a framework for the fine-mapping and ultimate cloning of a novel vascular gene at 8q22.3 or 14q13.

Fig. 1

(A) A metaphase spread prepared from blood samples of a patient with Klippel-Trenaunay syndrome (KTS002) and stained by Giemsa banding. A translocation involving chromosomes 8 and 14 (indicated by arrows) was identified. (B, C) Translocation t(8;14)(q22.3;q13) with centromeres as the solid line and breakpoints marked. Ideograms of normal chromosomes 8 and 14 and derivative chromosomes 8 and 14 are shown at the left (B), and G-banded chromosomes are shown at the right (C).

Fig. 2

Translocation t(8;14)(q22.3;q13) arose de novo. Pedigree structure of the KTS family with translocation t(8;14)(q22.3;q13). Results from clinical diagnosis of KTS and cytogenetic studies are summarized below each individual. Normal individuals are indicated as empty squares (males) or circles (females), and the affected individual is shown as a filled symbol. KTS, Klippel-Trenaunay syndrome; t(8;14), balanced translocation involving chromosomes 8q22.3; and 14q13.

Fig. 3

FISH mapping of the angiopoietin-1 gene (ANGPT1) to the vicinity of the 8q22.3 translocation breakpoint. (A–C). Examples of metaphase chromosomes 8, 14, and their derivatives (d8; d14) hybridized with chromosome 8-specific PAC clones 212d10 (A), 224e4 (B), and 111i04 (C). All three PAC probes (biotin-dUTP labeled ) showed hybridization signals (green color; detected with FITC-conjugated avidin) on normal chromosome 8 as well as derivative chromosome 8, but not on derivative chromosome 14. Therefore, ANGPT1 is located proximal to the 8q22.3 breakpoint. PAC 212d10 was then labeled with digoxigenin-dUTP and used in two-color FISH analysis (red signals detected with a rhodamine-conjugated anti-digoxigenin antibody). Both the red and green signals were detected on normal chromosome 8 as well as derivative chromosome 8, but not on derivative chromosome 14. Therefore, ANGPT1 is not disrupted by the 8q22.3 breakpoint. (D). PAC clones specific to ANGPT1. PAC clone 212d10 is specific to the 5′UTR of ANGPT1, and PAC clones 224e4 and 111i04 are specific to 3′UTR.

Fig. 4

Refined FISH mapping of the t(8;14) breakpoints. (A, B). FISH analysis using YAC probes 899a3 (A) and 956e8 (B) from the chromosome 8q22.3 breakpoint region. Hybridization signals for YAC 899a3 were observed on the normal chromosome 8 (8) and derivative chromosome 8 (d8). For YAC clone 956e8, FISH signals were observed on the normal chromosome 8 and derivative chromosome 14 (d14), indicating that the chromosome 8q22.3 breakpoint is located between YAC 899a3 and YAC 956e8. (C, D). FISH analysis using YAC probes 772d1 (C) and 964b11 (D) from the chromosome 14q13 breakpoint region, respectively. FISH signals were detected on the normal chromosome 14 (14) and derivative chromosome 14 (d14) with YAC 772d1 and on the normal chromosome 14 and derivative chromosome 8 (d8) with YAC 964b11, localizing the chromosome 14q13 breakpoint between YAC 772d1 and YAC964b11. (Metaphase chromosomes appeared to be blue color after DAPI counterstaining.)

Fig. 5

Localization of the 8q22.3 breakpoint (A) and the 14q13 translocation breakpoint (B) by FISH analysis. YACs spanning chromosome regions 8q22.3 (A) and 14q13 (B) were identified by searching the Whitehead Institute/MIT Center for Genome Research YAC database, and purchased from Research Genetics. The “+” symbol indicates that positive FISH signals were obtained on the corresponding derivative chromosome for a specific YAC, and “–” indicates absence of FISH signals on the corresponding derivative chromosome. The 8q22.3 breakpoint is defined within a <5-cM region flanked by markers AFMA082TG9 and GATA25E10, and the 14q13 breakpoint is between STSs WI-6583 and D14S989, <1 cM. ANGPT1, the angiopoietin-1 gene.