Clinical variability and novel mutations in the NHEJ1 gene in patients with a Nijmegen breakage syndrome-like phenotype.

We have previously shown that mutations in the genes encoding DNA Ligase IV (LIGIV) and RAD50, involved in DNA repair by nonhomologous-end joining (NHEJ) and homologous recombination, respectively, lead to clinical and cellular features similar to those of Nijmegen Breakage Syndrome (NBS). Very recently, a new member of the NHEJ repair pathway, NHEJ1, was discovered, and mutations in patients with features resembling NBS were described. Here we report on five patients from four families of different ethnic origin with the NBS-like phenotype. Sequence analysis of the NHEJ1 gene in a patient of Spanish and in a patient of Turkish origin identified homozygous, previously reported mutations, c.168C>G (p.Arg57Gly) and c.532C>T (p.Arg178Ter), respectively. Two novel, paternally inherited truncating mutations, c.495dupA (p.Asp166ArgfsTer20) and c.526C>T (p.Arg176Ter) and two novel, maternal genomic deletions of 1.9 and 6.9 kb of the NHEJ1 gene, were found in a compound heterozygous state in two siblings of German origin and in one Malaysian patient, respectively. Our findings confirm that patients with NBS-like phenotypes may have mutations in the NHEJ1 gene including multiexon deletions, and show that considerable clinical variability could be observed even within the same family.

Interstitial deletion 2p11.2-p12: report of a patient with mental retardation and review of the literature.

Deletions of chromosome bands 2p11.2 and 2p12 are rare, and only six patients have been reported to date. Here, we report on a 5-year-old girl with an 11.4 Mb interstitial deletion of chromosome bands 2p11.2-p12 and the characterization of this deletion by high-resolution array CGH. The patient presented with mental retardation, microcephaly and short stature. Facial features included broad nasal bridge, frontal bossing and mild dolichocephaly. Phenotypic comparison with previously published patients failed to reveal a consistent clinical pattern apart from developmental delay/mental retardation, which is probably due to different sizes and/or positions of the individual deletions. Among the 40 known genes deleted in our patient is REEP1, haploinsufficiency of which causes autosomal dominant spastic paraplegia type 31 (SPG31, OMIM 610250). Additional patients with well-characterized deletions within 2p11.2 and 2p12 will be needed to determine the role of individual genes for the clinical manifestations.

High-throughput sequencing of microdissected chromosomal regions.

The linkage of disease gene mapping with DNA sequencing is an essential strategy for defining the genetic basis of a disease. New massively parallel sequencing procedures will greatly facilitate this process, although enrichment for the target region before sequencing remains necessary. For this step, various DNA capture approaches have been described that rely on sequence-defined probe sets. To avoid making assumptions on the sequences present in the targeted region, we accessed specific cytogenetic regions in preparation for next-generation sequencing. We directly microdissected the target region in metaphase chromosomes, amplified it by degenerate oligonucleotide-primed PCR, and obtained sufficient material of high quality for high-throughput sequencing. Sequence reads could be obtained from as few as six chromosomal fragments. The power of cytogenetic enrichment followed by next-generation sequencing is that it does not depend on earlier knowledge of sequences in the region being studied. Accordingly, this method is uniquely suited for situations in which the sequence of a reference region of the genome is not available, including population-specific or tumor rearrangements, as well as previously unsequenced genomic regions such as centromeres.