Complex Reorganization and Predominant Non-Homologous Repair Following Chromosomal Breakage in Karyotypically Balanced Germline Rearrangements and Transgenic Integration

We defined the genetic landscape of balanced chromosomal rearrangements at nucleotide resolution by sequencing 141 breakpoints from cytogenetically-interpreted translocations and inversions. We confirm that the recently described phenomenon of “chromothripsis” (massive chromosomal shattering and reorganization) is not unique to cancer cells but also occurs in the germline where it can resolve to a karyotypically balanced state with frequent inversions. We detected a high incidence of complex rearrangements (19.2%) and substantially less reliance on microhomology (31%) than previously observed in benign CNVs. We compared these results to experimentally-generated DNA breakage-repair by sequencing seven transgenic animals, and revealed extensive rearrangement of the transgene and host genome with similar complexity to human germline alterations. Inversion is the most common rearrangement, suggesting that a combined mechanism involving template switching and non-homologous repair mediates the formation of balanced complex rearrangements that are viable, stably replicated and transmitted unaltered to subsequent generations

Using population admixture to help complete maps of the human genome

Tens of millions of base pairs of euchromatic human genome sequence, including many protein-coding genes, have no known location in the human genome. We describe an approach for localizing the human genome's missing pieces by utilizing the patterns of genome sequence variation created by population admixture. We mapped the locations of 70 scaffolds spanning four million base pairs of the human genome's unplaced euchromatic sequence, including more than a dozen protein-coding genes, and identified eight large novel inter-chromosomal segmental duplications. We find that most of these sequences are hidden in the genome's heterochromatin, particularly its pericentromeric regions. Many cryptic, pericentromeric genes are expressed in RNA and have been maintained intact for millions of years while their expression patterns diverged from those of paralogous genes elsewhere in the genome. We describe how knowledge of the locations of these sequences can inform disease association and genome biology studies

Multi-ancestry GWAS of the electrocardiographic PR interval identifies 202 loci underlying cardiac conduction

The electrocardiographic PR interval reflects atrioventricular conduction, and is associated with conduction abnormalities, pacemaker implantation, atrial fibrillation (AF), and cardiovascular mortality. Here we report a multi-ancestry (N=293,051) genome-wide association meta-analysis for the PR interval, discovering 202 loci of which 141 have not previously been reported. Variants at identified loci increase the percentage of heritability explained, from 33.5% to 62.6%. We observe enrichment for cardiac muscle developmental/contractile and cytoskeletal genes, highlighting key regulation processes for atrioventricular conduction. Additionally, 8 loci not previously reported harbor genes underlying inherited arrhythmic syndromes and/or cardiomyopathies suggesting a role for these genes in cardiovascular pathology in the general population. We show that polygenic predisposition to PR interval duration is an endophenotype for cardiovascular disease, including distal conduction disease, AF, and atrioventricular pre-excitation. These findings advance our understanding of the polygenic basis of cardiac conduction, and the genetic relationship between PR interval duration and cardiovascular disease. On the electrocardiogram, the PR interval reflects conduction from the atria to ventricles and also serves as risk indicator of cardiovascular morbidity and mortality. Here, the authors perform genome-wide meta-analyses for PR interval in multiple ancestries and identify 141 previously unreported genetic loci.Peer reviewe

Femoral derotational osteotomy level does not effect resulting torsion.

PURPOSE:The purpose of this study was to assess the effect on femoral torsion by rotational osteotomies at three different levels as measured in 3D using both the mechanical and the anatomic axes. METHODS:Ten cadaveric lower extremities underwent femoral osteotomies perpendicular to the anatomic axis (AA) at three levels: subtrochanteric, mid-diaphyseal and supracondylar. Parallel pins were placed, one in each femur segment. Computed tomography (CT) was acquired in post-osteotomies neutral position, then post-external rotation of the femur at each osteotomy level. Femurs were returned to neutral rotation between imaging exams. Using 3D CT reconstructions, custom software calculated femoral torsion (angle between the femoral neck axis and the posterior condylar axis in the transverse plane) and pin angle between segments, reoriented to both the mechanical axis (MA) and the AA. Pin angle and torsion change were compared for the three osteotomy locations (regression analysis and ANOVA performed). RESULTS:Two specimens were omitted (inadequate imaging); the remaining eight donors were 55-90 years old (mean: 64 ± 15 years), CT confirmed no bony defects. All three levels of osteotomy demonstrated significant correlations between the amount of rotation at the osteotomy (pin angle change) and the resulting change in femoral torsion (R square range 0.658-0.847). No significant differences were found between osteotomy level in torsion (MA:p = 0.285, AA:p = 0.156) or in pin angle (MA:p = 0.756, AA:p = 0.753). CONCLUSIONS:Performing a corrective rotational osteotomy orthogonal to the AA achieves the desired effect on MA regardless of location. This suggests that a surgeon's osteotomy level choice may be based on other risks/benefits of the various techniques

Using population admixture to help complete maps of the human genome.

Tens of millions of base pairs of euchromatic human genome sequence, including many protein-coding genes, have no known location in the human genome. We describe an approach for localizing the human genome's missing pieces using the patterns of genome sequence variation created by population admixture. We mapped the locations of 70 scaffolds spanning 4 million base pairs of the human genome's unplaced euchromatic sequence, including more than a dozen protein-coding genes, and identified 8 new large interchromosomal segmental duplications. We find that most of these sequences are hidden in the genome's heterochromatin, particularly its pericentromeric regions. Many cryptic, pericentromeric genes are expressed at the RNA level and have been maintained intact for millions of years while their expression patterns diverged from those of paralogous genes elsewhere in the genome. We describe how knowledge of the locations of these sequences can inform disease association and genome biology studies

Haploinsufficiency of KDM6A is associated with severe psychomotor retardation, global growth restriction, seizures and cleft palate

We describe a female subject (DGAP100) with a 46,X,t(X;5)(p11.3;q35.3)inv(5)(q35.3q35.1)dn, severe psychomotor retardation with hypotonia, global postnatal growth restriction, microcephaly, globally reduced cerebral volume, seizures, facial dysmorphia and cleft palate. Fluorescence in situ hybridization and whole-genome sequencing demonstrated that the X chromosome breakpoint disrupts KDM6A in the second intron. No genes were directly disrupted on chromosome 5. KDM6A is a histone 3 lysine 27 demethylase and a histone 3 lysine 4 methyltransferase. Expression of KDM6A is significantly reduced in DGAP100 lymphoblastoid cells compared to control samples. We identified nine additional cases with neurodevelopmental delay and various other features consistent with the DGAP100 phenotype with copy number variation encompassing KDM6A from microarray databases. We evaluated haploinsufficiency of kdm6a in a zebrafish model. kdm6a is expressed in the pharyngeal arches and ethmoid plate of the developing zebrafish, while a kdm6a morpholino knockdown exhibited craniofacial defects. We conclude KDM6A dosage regulation is associated with severe and diverse structural defects and developmental abnormalities.status: publishe

Haploinsufficiency of KDM6A is associated with severe psychomotor retardation, global growth restriction, seizures and cleft palate.

We describe a female subject (DGAP100) with a 46,X,t(X;5)(p11.3;q35.3)inv(5)(q35.3q35.1)dn, severe psychomotor retardation with hypotonia, global postnatal growth restriction, microcephaly, globally reduced cerebral volume, seizures, facial dysmorphia and cleft palate. Fluorescence in situ hybridization and whole-genome sequencing demonstrated that the X chromosome breakpoint disrupts KDM6A in the second intron. No genes were directly disrupted on chromosome 5. KDM6A is a histone 3 lysine 27 demethylase and a histone 3 lysine 4 methyl-transferase. Expression of KDM6A is significantly reduced in DGAP100 lymphoblastoid cells compared to control samples. We identified nine additional cases with neurodevelopmental delay and various other features consistent with the DGAP100 phenotype with copy number variation encompassing KDM6A from microarray databases. We evaluated haploinsufficiency of kdm6a in a zebrafish model. kdm6a is expressed in the pharyngeal arches and ethmoid plate of the developing zebrafish, while a kdm6a morpholino knockdown exhibited craniofacial defects. We conclude KDM6A dosage regulation is associated with severe and diverse structural defects and developmental abnormalities

Variation in life expectancy and mortality by cause among neighbourhoods in King County, WA, USA, 1990–2014: a census tract-level analysis for the Global Burden of Disease Study 2015

Background: Health outcomes are known to vary at both the country and local levels, but trends in mortality across a detailed and comprehensive set of causes have not been previously described at a very local level. Life expectancy in King County, WA, USA, is in the 95th percentile among all counties in the USA. However, little is known about how life expectancy and mortality from different causes of death vary at a local, neighbourhood level within this county. In this analysis, we estimated life expectancy and cause-specific mortality within King County to describe spatial trends, quantify disparities in mortality, and assess the contribution of each cause of death to overall disparities in all-cause mortality. Methods: We applied established so-called garbage code redistribution algorithms and small area estimation methods to death registration data for King County to estimate life expectancy, cause-specific mortality rates, and years of life lost (YLL) rates from 152 causes of death for 397 census tracts from Jan 1, 1990, to Dec 31, 2014. We used the cause list developed for the Global Burden of Disease 2015 study for this analysis. Deaths were tabulated by age group, sex, census tract, and cause of death. We used Bayesian mixed-effects regression models to estimate mortality overall and from each cause. Findings: Between 1990 and 2014, life expectancy in King County increased by 5·4 years (95% uncertainty interval [UI] 5·0–5·7) among men (from 74·0 years [73·7–74·3] to 79·3 years [79·1–79·6]) and by 3·4 years (3·0–3·7) among women (from 80·0 years [79·7–80·2] to 83·3 years [83·1–83·5]). In 2014, life expectancy ranged from 68·4 years (95% UI 66·9–70·1) to 86·7 years (85·0–88·2) for men and from 73·6 years (71·6–75·5) to 88·4 years (86·9–89·9) for women among census tracts within King County. Rates of YLL by cause also varied substantially among census tracts for each cause of death. Geographical areas with relatively high and relatively low YLL rates differed by cause. In general, causes of death responsible for more YLLs overall also contributed more significantly to geographical inequality within King County. However, certain causes contributed more to inequality than to overall YLLs. Interpretation: This census tract-level analysis of life expectancy and cause-specific YLL rates highlights important differences in health among neighbourhoods in King County that are masked by county-level estimates. Efforts to improve population health in King County should focus on reducing geographical inequality, by targeting those health conditions that contribute the most to overall YLLs and to inequality. This analysis should be replicated in other locations to more fully describe fine-grained local-level variation in population health and contribute to efforts to improve health while reducing inequalities. Funding: John W Stanton and Theresa E Gillespie

Next-Generation Sequencing Strategies Enable Routine Detection of Balanced Chromosome Rearrangements for Clinical Diagnostics and Genetic Research

The contribution of balanced chromosomal rearrangements to complex disorders remains unclear because they are not detected routinely by genome-wide microarrays and clinical localization is imprecise. Failure to consider these events bypasses a potentially powerful complement to single nucleotide polymorphism and copy-number association approaches to complex disorders, where much of the heritability remains unexplained. To capitalize on this genetic resource, we have applied optimized sequencing and analysis strategies to test whether these potentially high-impact variants can be mapped at reasonable cost and throughput. By using a whole-genome multiplexing strategy, rearrangement breakpoints could be delineated at a fraction of the cost of standard sequencing. For rearrangements already mapped regionally by karyotyping and fluorescence in situ hybridization, a targeted approach enabled capture and sequencing of multiple breakpoints simultaneously. Importantly, this strategy permitted capture and unique alignment of up to 97% of repeat-masked sequences in the targeted regions. Genome-wide analyses estimate that only 3.7% of bases should be routinely omitted from genomic DNA capture experiments. Illustrating the power of these approaches, the rearrangement breakpoints were rapidly defined to base pair resolution and revealed unexpected sequence complexity, such as co-occurrence of inversion and translocation as an underlying feature of karyotypically balanced alterations. These findings have implications ranging from genome annotation to de novo assemblies and could enable sequencing screens for structural variations at a cost comparable to that of microarrays in standard clinical practice