Accurate Breakpoint Mapping in Apparently Balanced Translocation Families with Discordant Phenotypes Using Whole Genome Mate-Pair Sequencing

<div><p>Familial apparently balanced translocations (ABTs) segregating with discordant phenotypes are extremely challenging for interpretation and counseling due to the scarcity of publications and lack of routine techniques for quick investigation. Recently, next generation sequencing has emerged as an efficacious methodology for precise detection of translocation breakpoints. However, studies so far have mainly focused on <i>de novo</i> translocations. The present study focuses specifically on familial cases in order to shed some light to this diagnostic dilemma. Whole-genome mate-pair sequencing (WG-MPS) was applied to map the breakpoints in nine two-way ABT carriers from four families. Translocation breakpoints and patient-specific structural variants were validated by Sanger sequencing and quantitative Real Time PCR, respectively. Identical sequencing patterns and breakpoints were identified in affected and non-affected members carrying the same translocations. <i>PTCD1</i>, <i>ATP5J2-PTCD1</i>, <i>CADPS2</i>, and <i>STPG1</i> were disrupted by the translocations in three families, rendering them initially as possible disease candidate genes. However, subsequent mutation screening and structural variant analysis did not reveal any pathogenic mutations or unique variants in the affected individuals that could explain the phenotypic differences between carriers of the same translocations. In conclusion, we suggest that NGS-based methods, such as WG-MPS, can be successfully used for detailed mapping of translocation breakpoints, which can also be used in routine clinical investigation of ABT cases. Unlike <i>de novo</i> translocations, no associations were determined here between familial two-way ABTs and the phenotype of the affected members, in which the presence of cryptic imbalances and complex chromosomal rearrangements has been excluded. Future whole-exome or whole-genome sequencing will potentially reveal unidentified mutations in the patients underlying the discordant phenotypes within each family. In addition, larger studies are needed to determine the exact percentage for phenotypic risk in families with ABTs.</p></div

Family 1 results with t(1;7) translocation.

<p>A) Family 1 pedigree showing the proband with intellectual disability, psychomotor delay and epilepsy, as well as the non-affected mother and father. The t(1;7)(p36.1;q22) translocation is maternally inherited. B) Ideograms showing the normal and derivative (der) chromosomes (chr) 1 and 7 (not to scale). Genetic material from chr1 and chr7 is shown with a solid, orange and dotted, blue frame line, respectively. The approximate breakpoint positions on 1p36.1 and 7q22 are indicated by arrows. C) UPD7 analysis results from one of the informative microsatellite markers (D71824) in the affected proband and non-affected parents; by comparing the peak sizes between all family members, normal biparental inheritance was concluded. D) Quantitative Real-Time PCR results demonstrating the paternal inheritance of the chr3 duplication predicted from the structural variant analysis in the affected proband.</p

Translocation junction sequences identified in each family.

<p>Translocation junction sequences to the base-pair level as identified by mate-pair sequencing and verified by Sanger sequencing in A) family 1 with t(1;7)(p36.1;q22), B) family 2 with t(7;8)(q32;q24.13), C) family 3 with t(4;10)(q35;q11.2), and D) family 4 with t(1;20)(p35.3;q13.3). Translocation junction sequences (middle line) and matching reference sequences (top and bottom lines) are shown with a different colour depending on the chromosome involved (chr1-orange; chr4-purple; chr7-blue; chr8-red; chr10-yellow; chr20-green). Microhomology observed at the translocation breakpoint sites is highlighted in yellow, deleted sequences around the breakpoints are underlined, duplicated sequences are in bold, capital letters, and inserted sequences not aligning to either chromosome are in bold, lower-case letters.</p

Breakpoint mapping and sequencing results for each apparently balanced translocation case included in this study.

<p>Breakpoint mapping and sequencing results for each apparently balanced translocation case included in this study.</p

The <i>ZNF423</i> gene and quantitative Real-Time PCR (qRT-PCR) results.

<p>A) Schematic illustration of the <i>ZNF423</i> gene (minus strand). The protein-coding exons and introns of the gene are represented with solid, vertical lines and dotted, horizontal lines, respectively (not to scale). Intron 3 is enlarged to demonstrate the approximate <i>ZNF423</i> deletion position (blue line) as predicted by MPS in the affected sibling in family 2, as well as the amplicon (Z-RT1, Z-RT2, and Z-RT3) positions (red lines) from the qRT-PCR analysis. The actual coordinates are given on the left (GRCh37/hg19). B) qRT-PCR results demonstrating a reduced relative <i>ZNF423</i> copy number in the proband and normal results in the non-affected sibling as compared with a control. Parental genomic material was unavailable and thus not included in the analysis.</p