Delineation of molecular bases of chromosomal and Mendelian phenotypes

Translocations are a common class of chromosomal aberrations and can cause disease by physically disrupting genes or altering their regulatory environment. Some translocations, apparently balanced at the microscopic level, demonstrate deletions, duplications, insertions, or inversions at the molecular level. Traditionally, chromosomal rearrangements have been investigated with a conventional banded karyotype followed by arduous positional cloning projects. More recently, molecular cytogenetic approaches using fluorescence in situ hybridization (FISH), array comparative genomic hybridization (aCGH), or whole-genome SNP genotyping together with molecular methods such as inverse PCR and quantitative PCR have allowed more precise evaluation of the breakpoints. These methods suffer, however, from being experimentally intensive, time-consuming and of less than single base pair resolution. Here we describe targeted breakpoint capture followed by next-generation sequencing (TBCS) as a new approach to the general problem of determining the precise structural characterization of translocation breakpoints and related chromosomal aberrations. We tested this approach in three patients with complex chromosomal translocations: The first has craniofacial abnormalities and an apparently balanced t(2;3)(p15;q12) translocation; the second has cleidocranial dysplasia (OMIM 119600) associated with a t(2;6)(q22;p12.3) translocation and a breakpoint in RUNX2 on chromosome 6p; and the third has acampomelic campomelic dysplasia (OMIM 114290) associated with a t(5;17)(q23.2;q24) translocation, with a breakpoint centromeric to SOX9 on chromosome 17q. Preliminary studies indicated complex rearrangements in patients 1 and 3 with a total of 10 predicted breakpoints in the three patients. Using TBCS, we quickly and precisely defined eight of the 10 breakpoints. We were unable to define the 2 breakpoints from the 2;6 translocation because we designed baits only to the 640 kb predicted breakpoint region on chromosome 6, a region rich in DNA repeats that was not captured. We elected not to capture the breakpoint region on chromosome 2 because our preliminary localization of the breakpoint was imprecise with a minimal region of 4.9 Mb.;Next-generation sequencing is rapidly becoming a potent technology to identify the molecular basis of Mendelian disorders. Currently more than 2,779 genes have been shown to contain variants that cause Mendelian disease, but there are still several thousand phenotypes yet to be molecularly defined. The ability of new whole-genome sequencing (WGS) and whole-exome sequencing (WES) technologies to expose most of the genetic variants in any given genome opens an exciting opportunity to identify the responsible disease genes. The major challenge is to determine which of the large number of variants from the reference sequence that each person carries is responsible for the Mendelian phenotype. In many instances genetic methods can be combined with genome wide sequencing (either WGS or WES) to point to the responsible variant. To test this idea, we sequenced the whole genome of a single patient with an unexplained dominant Mendelian disease, metachondromatosis (OMIM 156250), and used partial linkage data from her small family to focus our search for the responsible variant. In the proband, we identified an 11 bp deletion in exon four of PTPN11, which alters frame, results in premature translation termination, and co-segregates with the phenotype. We confirmed our result in a second metachondromatosis family by identifying a nonsense mutation in exon 4 of PTPN11 that also co-segregated with the phenotype. Sequencing PTPN11 exon 4 in 469 controls showed no such protein truncating variants, supporting the pathogenicity of these two mutations.;In a second family we performed WES in the proband with an undiagnosed disorder and his unaffected brother who were products of a consanguineous union. We identified a homozygous 17 by deletion in exon 4 of SCARF2 in the proband that was not present in his unaffected brother and led us to the diagnosis of Van den Ende–Gupta Syndrome (VDEGS) (OMIM - 600920). Interestingly, this patient has severe bilateral sclerocornea, a feature previously described in only one of 25 reported VDEGS patients and thought to be secondary to an associated 22q11.2 microdeletion. Our patient expands the VDEGS phenotype and implicates SCARF2 as the causative gene of sclerocornea in these patients.;We conclude that the combination of a new technology (next-generation sequencing) and classical genetic approachs provides a powerful strategy to determining the precise structural characterization of translocation breakpoints and related chromosomal aberrations and to discover the genes responsible for unexplained Mendelian disorders.