Background The UK 100,000 Genomes Project is in the process of investigating the role of genome sequencing of patients with undiagnosed rare disease following usual care, and the alignment of research with healthcare implementation in the UK’s national health service. (Other parts of this Project focus on patients with cancer and infection.) Methods We enrolled participants, collected clinical features with human phenotype ontology terms, undertook genome sequencing and applied automated variant prioritization based on virtual gene panels (PanelApp) and phenotypes (Exomiser), alongside identification of novel pathogenic variants through research analysis. We report results on a pilot study of 4660 participants from 2183 families with 161 disorders covering a broad spectrum of rare disease. Results Diagnostic yields varied by family structure and were highest in trios and larger pedigrees. Likely monogenic disorders had much higher diagnostic yields (35%) with intellectual disability, hearing and vision disorders, achieving yields between 40 and 55%. Those with more complex etiologies had an overall 25% yield. Combining research and automated approaches was critical to 14% of diagnoses in which we found etiologic non-coding, structural and mitochondrial genome variants and coding variants poorly covered by exome sequencing. Cohort-wide burden testing across 57,000 genomes enabled discovery of 3 new disease genes and 19 novel associations. Of the genetic diagnoses that we made, 24% had immediate ramifications for the clinical decision-making for the patient or their relatives. Conclusion Our pilot study of genome sequencing in a national health care system demonstrates diagnostic uplift across a range of rare diseases. (Funded by National Institute for Health Research and others)

22q11.2 microduplications of a 3-Mb region surrounded by low-copy repeats should be, theoretically, as frequent as the deletions of this region; however, few microduplications have been reported. We show that the phenotype of these patients with microduplications is extremely diverse, ranging from normal to behavioral abnormalities to multiple defects, only some of which are reminiscent of the 22q11.2 deletion syndrome. This diversity will make ascertainment difficult and will necessitate a rapid-screening method. We demonstrate the utility of four different screening methods. Although all the screening techniques give unique information, the efficiency of real-time polymerase chain reaction allowed the discovery of two 22q11.2 microduplications in a series of 275 females who tested negative for fragile X syndrome, thus widening the phenotypic diversity. Ascertainment of the fragile X-negative cohort was twice that of the cohort screened for the 22q11.2 deletion. We also report the first patient with a 22q11.2 triplication and show that this patient's mother carries a 22q11.2 microduplication. We strongly recommend that other family members of patients with 22q11.2 microduplications also be tested, since we found several phenotypically normal parents who were carriers of the chromosomal abnormality.

Low-copy repeats, or segmental duplications, are highly dynamic regions in the genome. The low-copy repeats on chromosome 22q11.2 (LCR22) are a complex mosaic of genes and pseudogenes formed by duplication processes; they mediate chromosome rearrangements associated with velo-cardio-facial syndrome/DiGeorge syndrome, der (22) syndrome, and cat-eye syndrome. The ability to trace the substrates and products of recombination events provides a unique opportunity to identify the mechanisms responsible for shaping LCR22s. We examined the genomic sequence of known LCR22 genes and their duplicated derivatives. We found Alu (SINE) elements at the breakpoints in the substrates and at the junctions in the truncated products of recombination for USP18, GGT, and GGTLA, consistent with Alu-mediated unequal crossing-over events. In addition, we were able to trace a likely interchromosomal Alu-mediated fusion between IGSF3 on 1p13.1 and GGT on 22q11.2. Breakpoints occurred inside Alu elements as well as in the 5Ј or 3Ј ends of them. A possible stimulus for the 5Ј or 3Ј terminal rearrangements may be the high sequence similarities between different Alu elements, combined with a potential recombinogenic role of retrotransposon target-site duplications flanking the Alu element, containing potentially kinkable DNA sites. Such sites may represent focal points for recombination. Thus, genome shuffling by Alu-mediated rearrangements has contributed to genome architecture during primate evolution.

Interstitial deletions of 6q are associated with variable phenotypes, including growth retardation, dysmorphic features, upper limb malformations, and Prader-Willi (PW)-like features. Only a minority of cases in the literature have been characterized with high resolution techniques, making genotype-phenotype correlations difficult. We report 12 individuals with overlapping, 200-kb to 16.4-Mb interstitial deletions within 6q15q22.33 characterized by microarray-based comparative genomic hybridization to better correlate deletion regions with specific phenotypes. Four individuals have a PW-like phenotype, though only two have deletion of SIM1, the candidate gene for this feature. Therefore, other genes on 6q may contribute to this phenotype including multiple genes on 6q16 and our newly proposed candidate, the transcription cofactor gene VGLL2 on 6q22.2. Two individuals present with movement disorders as a major feature, and ataxia is present in a third. The 4.1-Mb 6q22.1q22.2 critical region for movement disorders includes the cerebellar-expressed candidate gene GOPC. Observed brain malformations include thick corpus callosum in two subjects, cerebellar vermal hypoplasia in two subjects, and cerebellar atrophy in one subject. Seven subjects' deletions overlap a ~250-kb cluster of four genes on 6q22.1 including MARCKS, HDAC2, and HS3ST5, which are involved in neural development. Two subjects have only this gene cluster deleted, and one deletion was apparently de novo, suggesting at least one of these genes plays an important role in development. Although the phenotypes associated with 6q deletions can vary, using overlapping deletions to delineate critical regions improves genotype-phenotype correlation for interstitial 6q deletions.

The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions ('Döhle-like' bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8-10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.