Target-enrichment strategies for next-generation sequencing.

We have not yet reached a point at which routine sequencing of large numbers of whole eukaryotic genomes is feasible, and so it is often necessary to select genomic regions of interest and to enrich these regions before sequencing. There are several enrichment approaches, each with unique advantages and disadvantages. Here we describe our experiences with the leading target-enrichment technologies, the optimizations that we have performed and typical results that can be obtained using each. We also provide detailed protocols for each technology so that end users can find the best compromise between sensitivity, specificity and uniformity for their particular project.

Early Diagnosis of Werner's Syndrome Using Exome-Wide Sequencing in a Single, Atypical Patient.

Genetic diagnosis of inherited metabolic disease is conventionally achieved through syndrome recognition and targeted gene sequencing, but many patients receive no specific diagnosis. Next-generation sequencing allied to capture of expressed sequences from genomic DNA now offers a powerful new diagnostic approach. Barriers to routine diagnostic use include cost, and the complexity of interpreting results arising from simultaneous identification of large numbers of variants. We applied exome-wide sequencing to an individual, 16-year-old daughter of consanguineous parents with a novel syndrome of short stature, severe insulin resistance, ptosis, and microcephaly. Pulldown of expressed sequences from genomic DNA followed by massively parallel sequencing was undertaken. Single nucleotide variants were called using SAMtools prior to filtering based on sequence quality and existence in control genomes and exomes. Of 485 genetic variants predicted to alter protein sequence and absent from control data, 24 were homozygous in the patient. One mutation - the p.Arg732X mutation in the WRN gene - has previously been reported in Werner's syndrome (WS). On re-evaluation of the patient several early features of WS were detected including loss of fat from the extremities and frontal hair thinning. Lymphoblastoid cells from the proband exhibited a defective decatenation checkpoint, consistent with loss of WRN activity. We have thus diagnosed WS some 15 years earlier than average, permitting aggressive prophylactic therapy and screening for WS complications, illustrating the potential of exome-wide sequencing to achieve early diagnosis and change management of rare autosomal recessive disease, even in individual patients of consanguineous parentage with apparently novel syndromes.

Clustered coding variants in the glutamate receptor complexes of individuals with schizophrenia and bipolar disorder.

Current models of schizophrenia and bipolar disorder implicate multiple genes, however their biological relationships remain elusive. To test the genetic role of glutamate receptors and their interacting scaffold proteins, the exons of ten glutamatergic 'hub' genes in 1304 individuals were re-sequenced in case and control samples. No significant difference in the overall number of non-synonymous single nucleotide polymorphisms (nsSNPs) was observed between cases and controls. However, cluster analysis of nsSNPs identified two exons encoding the cysteine-rich domain and first transmembrane helix of GRM1 as a risk locus with five mutations highly enriched within these domains. A new splice variant lacking the transmembrane GPCR domain of GRM1 was discovered in the human brain and the GRM1 mutation cluster could perturb the regulation of this variant. The predicted effect on individuals harbouring multiple mutations distributed in their ten hub genes was also examined. Diseased individuals possessed an increased load of deleteriousness from multiple concurrent rare and common coding variants. Together, these data suggest a disease model in which the interplay of compound genetic coding variants, distributed among glutamate receptors and their interacting proteins, contribute to the pathogenesis of schizophrenia and bipolar disorders.

Esophageal atresia, hypoplasia of zygomatic complex, microcephaly, cup-shaped ears, congenital heart defect, and mental retardation--new MCA/MR syndrome in two affected sibs and a mildly affected mother?

The previously undescribed combination of esophageal atresia, hypoplasia of the zygomatic complex, microcephaly, cup-shaped ears, congenital heart defect, and mental retardation was diagnosed in two siblings of different sexes, with the brother being more severely affected. The mother presented with zygomatic arch hypoplasia of the right side only. We discuss major differential diagnoses: Goldenhar, Feingold, CHARGE, and Treacher Collins syndromes show a few overlapping clinical features, but these diagnoses are unlikely as the clinical findings are unusual for Goldenhar syndrome and mutational screening of the MYCN, the CHD7, and the TCOF1 genes did not reveal any abnormalities. Autosomal recessive oto-facial syndrome, hypomandibular faciocranial dysostosis, and Ozkan syndromes were clinically excluded. A microdeletion 22q11.2 was excluded by FISH analysis, a microdeletion 2p23-p24 by microsatellite analyses, a subtelomeric chromosomal aberration by MLPA, and a small genomic deletion/duplication by CGH array. As X-inactivation studies did not show skewed X-inactivation in the mother, we consider X-chromosomal recessive inheritance of this condition less likely. We discuss autosomal dominant inheritance with variable expressivity or mosaicism in the mother as the likely genetic mechanism in this new multiple congenital anomaly/mental retardation (MCA/MR) syndrome.