[Show abstract][Hide abstract] ABSTRACT: Purpose:
Age-related macular degeneration (AMD) has a substantial genetic risk component, as evidenced by the risk from common genetic variants uncovered in the first genome-wide association studies. More recently, it has become apparent that rare genetic variants also play an independent role in AMD risk. We sought to determine if rare variants in complement factor H (CFH) played a role in AMD risk.
We had previously collected DNA from a large population of patients with advanced age-related macular degeneration (A-AMD) and controls for targeted deep sequencing of candidate AMD risk genes. In this analysis, we tested for an increased burden of rare variants in CFH in 1665 cases and 752 controls from this cohort.
We identified 65 missense, nonsense, or splice-site mutations with a minor allele frequency ≤ 1%. Rare variants with minor allele frequency ≤ 1% (odds ratio [OR] = 1.5, P = 4.4 × 10-2), 0.5% (OR = 1.6, P = 2.6 × 10-2), and all singletons (OR = 2.3, P = 3.3 × 10-2) were enriched in A-AMD cases. Moreover, we observed loss-of-function rare variants (nonsense, splice-site, and loss of a conserved cysteine) in 10 cases and serum levels of FH were decreased in all 5 with an available sample (haploinsufficiency). Further, rare variants in the major functional domains of CFH were increased in cases (OR = 3.2; P = 1.4 × 10-3) and the magnitude of the effect correlated with the disruptive nature of the variant, location in an active site, and inversely with minor allele frequency.
In this large A-AMD cohort, rare variants in the CFH gene were enriched and tended to be located in functional sites or led to low serum levels. These data, combined with those indicating a similar, but even more striking, increase in rare variants found in CFI, strongly implicate complement activation in A-AMD etiopathogenesis as CFH and CFI interact to inhibit the alternative pathway.
[Show abstract][Hide abstract] ABSTRACT: Germline, loss-of-function mutations in the transcription factor STAT3 cause immunodeficiency, while somatic, gain-of-function mutations in STAT3 are associated with large granular lymphocytic leukemic, myelodysplastic syndrome, and aplastic anemia. Recently, germline mutations in STAT3 have also been associated with autoimmune disease. Here we report thirteen individuals from ten families with lymphoproliferation and early-onset, solid organ autoimmunity associated with nine different germline, heterozygous mutations in STAT3. Patients exhibited a variety of clinical features, with most having lymphadenopathy, autoimmune cytopenias, multi-organ autoimmunity (lung, gastrointestinal, hepatic, and/or endocrine dysfunction), infections and short stature. Functional analyses demonstrate that these mutations confer a gain-of-function in STAT3 leading to secondary defects in STAT5 and STAT1 phosphorylation and the regulatory T cell compartment. Treatment targeting a cytokine pathway that signals through STAT3 lead to clinical improvement in one patient, suggesting a potential therapeutic option for such patients. These results suggest that there is a broad range of autoimmunity caused by germline STAT3 gain-of-function mutations, and that hematologic autoimmunity is a major component of this newly-described disorder. Some patients for this study were enrolled in a trial registered at ClinicalTrials.gov #NCT00001350.
[Show abstract][Hide abstract] ABSTRACT: Uveal melanoma is the most common primary cancer of the eye and often results in fatal metastasis. Here, we describe mutations occurring exclusively at codon 625 of the SF3B1 gene, encoding splicing factor 3B subunit 1, in low-grade uveal melanomas with good prognosis. Thus, uveal melanoma is among a small group of cancers associated with SF3B1 mutations, and these mutations denote a distinct molecular subset of uveal melanomas.
[Show abstract][Hide abstract] ABSTRACT: Psoriasis is a common, immune-mediated genetic disorder of the skin and is associated with arthritis in approximately 30% of cases. Previously, we localized PSORS2 (psoriasis susceptibility locus 2) to chromosomal region 17q25.3-qter after a genome-wide linkage scan in a family of European ancestry with multiple cases of psoriasis and psoriatic arthritis. Linkage to PSORS2 was also observed in a Taiwanese family with multiple psoriasis-affected members. In caspase recruitment domain family, member 14 (CARD14), we identified unique gain-of-function mutations that segregated with psoriasis by using genomic capture and DNA sequencing. The mutations c.349G>A (p.Gly117Ser) (in the family of European descent) and c.349+5G>A (in the Taiwanese family) altered splicing between CARD14 exons 3 and 4. A de novo CARD14 mutation, c.413A>C (p.Glu138Ala), was detected in a child with sporadic, early-onset, generalized pustular psoriasis. CARD14 activates nuclear factor kappa B (NF-kB), and compared with wild-type CARD14, the p.Gly117Ser and p.Glu138Ala substitutions were shown to lead to enhanced NF-kB activation and upregulation of a subset of psoriasis-associated genes in keratinocytes. These genes included chemokine (C-C motif) ligand 20 (CCL20) and interleukin 8 (IL8). CARD14 is localized mainly in the basal and suprabasal layers of healthy skin epidermis, whereas in lesional psoriatic skin, it is reduced in the basal layer and more diffusely upregulated in the suprabasal layers of the epidermis. We propose that, after a triggering event that can include epidermal injury, rare gain-of-function mutations in CARD14 initiate a process that includes inflammatory cell recruitment by keratinocytes. This perpetuates a vicious cycle of epidermal inflammation and regeneration, a cycle which is the hallmark of psoriasis.
Full-text · Article · Apr 2012 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: Psoriasis is a common inflammatory disorder of the skin and other organs. We have determined that mutations in CARD14, encoding a nuclear factor of kappa light chain enhancer in B cells (NF-kB) activator within skin epidermis, account for PSORS2. Here, we describe fifteen additional rare missense variants in CARD14, their distribution in seven psoriasis cohorts (>6,000 cases and >4,000 controls), and their effects on NF-kB activation and the transcriptome of keratinocytes. There were more CARD14 rare variants in cases than in controls (burden test p value = 0.0015). Some variants were only seen in a single case, and these included putative pathogenic mutations (c.424G>A [p.Glu142Lys] and c.425A>G [p.Glu142Gly]) and the generalized-pustular-psoriasis mutation, c.413A>C (p.Glu138Ala); these three mutations lie within the coiled-coil domain of CARD14. The c.349G>A (p.Gly117Ser) familial-psoriasis mutation was present at a frequency of 0.0005 in cases of European ancestry. CARD14 variants led to a range of NF-kB activities; in particular, putative pathogenic variants led to levels >2.5× higher than did wild-type CARD14. Two variants (c.511C>A [p.His171Asn] and c.536G>A [p.Arg179His]) required stimulation with tumor necrosis factor alpha (TNF-α) to achieve significant increases in NF-kB levels. Transcriptome profiling of wild-type and variant CARD14 transfectants in keratinocytes differentiated probably pathogenic mutations from neutral variants such as polymorphisms. Over 20 CARD14 polymorphisms were also genotyped, and meta-analysis revealed an association between psoriasis and rs11652075 (c.2458C>T [p.Arg820Trp]; p value = 2.1 × 10(-6)). In the two largest psoriasis cohorts, evidence for association increased when rs11652075 was conditioned on HLA-Cw*0602 (PSORS1). These studies contribute to our understanding of the genetic basis of psoriasis and illustrate the challenges faced in identifying pathogenic variants in common disease.
Full-text · Article · Apr 2012 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: Paroxysmal kinesigenic dyskinesia with infantile convulsions (PKD/IC) is an episodic movement disorder with autosomal-dominant inheritance and high penetrance, but the causative genetic mutation is unknown. We have now identified four truncating mutations involving the gene PRRT2 in the vast majority (24/25) of well-characterized families with PKD/IC. PRRT2 truncating mutations were also detected in 28 of 78 additional families. PRRT2 encodes a proline-rich transmembrane protein of unknown function that has been reported to interact with the t-SNARE, SNAP25. PRRT2 localizes to axons but not to dendritic processes in primary neuronal culture, and mutants associated with PKD/IC lead to dramatically reduced PRRT2 levels, leading ultimately to neuronal hyperexcitability that manifests in vivo as PKD/IC.
[Show abstract][Hide abstract] ABSTRACT: A set of Centre d'Étude du Polymorphisme Humain (CEPH) cell lines serves as a large reference collection that has been widely used as a benchmark for allele frequencies in the analysis of genetic variants, to create linkage maps of the human genome, to study the genetics of gene expression, to provide samples to the HapMap and 1000 Genomes projects, and for a variety of other applications. An explicit feature of the CEPH collection is that these multigenerational families represent reference panels of known relatedness, consisting mostly of three-generation pedigrees with large sibships, two parents, and grandparents. We applied identity-by-state (IBS) and identity-by-descent (IBD) methods to high-density genotype data from 186 CEPH individuals in 13 families. We identified unexpected relatedness between nominally unrelated grandparents both within and between pedigrees. For one pair, the estimated Cotterman coefficient of relatedness k1 exceeded 0.2, consistent with one-eighth sharing (eg, first-cousins). Unexpectedly, significant IBD2 values were discovered in both second-degree and parent-child relationships. These were accompanied by regions of homozygosity in the offspring, which corresponded to blocks lacking IBS0 in purportedly unrelated parents, consistent with inbreeding. Our findings support and extend a 1999 report, based on the use of short tandem-repeat polymorphisms, that several CEPH families had regions of homozygosity consistent with autozygosity. We benchmarked our IBD approach (called kcoeff) against both RELPAIR and PREST software packages. Our findings may affect the interpretation of previous studies and the design of future studies that rely on the CEPH resource.
Full-text · Article · Jan 2012 · European journal of human genetics: EJHG
[Show abstract][Hide abstract] ABSTRACT: Paroxysmal Kinesigenic Dyskinesia with Infantile Convulsions (PKD/IC) is an episodic movement disorder with autosomal dominant inheritance and high penetrance, but the causative gene is unknown. We have now identified four truncating mutations involving the PRRT2 gene in the vast majority (24/25) of well characterized families with PKD/IC. PRRT2 truncating mutations were also detected in 28 of 78 additional families. The PRRT2 gene encodes a proline-rich transmembrane protein of unknown function that has been reported to interact with the t-SNARE, SNAP25. PRRT2 localizes to axons but not to dendritic processes in primary neuronal culture and mutants associated with PKD/IC lead to dramatically reduced PRRT2 protein levels leading ultimately to neuronal hyperexcitability that manifests in vivo as PKD/IC.
[Show abstract][Hide abstract] ABSTRACT: Psoriasis is a chronic inflammatory immune-mediated disorder affecting the skin and other organs including joints. Over 1,300 transcripts are altered in psoriatic involved skin compared with normal skin. However, to our knowledge, global epigenetic profiling of psoriatic skin is previously unreported. Here, we describe a genome-wide study of altered CpG methylation in psoriatic skin. We determined the methylation levels at 27,578 CpG sites in skin samples from individuals with psoriasis (12 involved, 8 uninvolved) and 10 unaffected individuals. CpG methylation of involved skin differed from normal skin at 1,108 sites. Twelve mapped to the epidermal differentiation complex, upstream or within genes that are highly upregulated in psoriasis. Hierarchical clustering of 50 of the top differentially methylated (DM) sites separated psoriatic from normal skin samples with uninvolved skin exhibiting intermediate methylation. CpG sites where methylation was correlated with gene expression are reported. Sites with inverse correlations between methylation and nearby gene expression include those of KYNU, OAS2, S100A12, and SERPINB3, whose strong transcriptional upregulation is an important discriminator of psoriasis. Pyrosequencing of bisulfite-treated DNA from skin biopsies at three DM loci confirmed earlier findings and revealed reversion of methylation levels toward the non-psoriatic state after 1 month of anti-TNF-α therapy.
Full-text · Article · Nov 2011 · Journal of Investigative Dermatology
[Show abstract][Hide abstract] ABSTRACT: Quality control information for each ethnic group. Quality control was performed using PLINK to remove individuals with ≤98% genotype call rate, SNPs that failed a HWE (p <0.0001), SNPs that had ≤99% genotype call rate, and SNPS with a MAF ≤0.01. The first MEX, CHI, CAU, and AA columns represent the quality control measures in which individuals with low genotyping rates were removed. The second MEX, CHI, CAU, and AA columns represent the quality control measures in which individuals with low genotyping rates were not removed.
[Show abstract][Hide abstract] ABSTRACT: Evidence for apparent IBD0 sharing between previously annotated parent-child relationships. For two pairs of related individuals who were previously annotated parent-child, we show IBD0 sharing across various chromosomes as provided by SNPduo analysis. For each pairwise comparison the three tracks are IBS0, IBS1, and IBS2. We also show the genotypes of the individuals, which indicate the individual who has the genotype profile that leads to the measured IBS0. For each individual the genotype tracks are BB, AB, AA, and NC (missing genotype). (A) Previously annotated parent-child relationship between CEU members NA12874 (maternal grandfather) and NA12865 (mother) has apparent IBD0 across chromosome 1. (B) Genotypes of NA12874, which reveal considerable homozygosity across the q arm. Note the IBS0 in this region. (C) Genotypes of NA12865, which are normal across the entire chromosome. (D) Ideogram of chromosome 1. (E) Previously annotated parent-child relationship between YRI members NA18498 (father) and NA18497 (son) across chromosome 1 has apparent IBD0. (F) Genotypes of NA18497 in which a region of dense NCs overlaps a region lacking AB calls in the same region of IBS0 between the parent-child relationship. (G) Genotypes of NA18498, which are normal across the entire chromosome. (H) Ideogram of chromosome 1.
[Show abstract][Hide abstract] ABSTRACT: Overview of the IBD method. Upper panels: representative IBS observations between two related individuals across a typical chromosomal segment (x-axis). Each data point corresponds to a single locus for which IBS0, IBS1, or IBS2 is observed and plotted (y-axis). We delineate four regions using an iterative approach (typically using 300 informative SNPs per window). In Region 1, having IBS0, we detect IBS0 and infer an IBD0 region. In Region 2, lacking IBS0 calls, we define the locus as “not IBD0.” We infer the IBD1 versus IBD2 state depending on the IBS1 calls (based on allele frequency estimator c). Region 3, having observed IBS1 calls, is inferred to be IBD1. Region 4, lacking observed IBS1 calls, is inferred to be IBD2. K0, K1, and K2 estimates are defined in the figure and correspond to Cotterman coefficients of relatedness k0, k1, and k2, respectively.
[Show abstract][Hide abstract] ABSTRACT: IBD estimates for previously annotated and novel relationships. We report the IBD estimates for every pairwise comparison that we report as related within HapMap Phase III release 3 (n = 2,261). This includes previously annotated relationships (denoted by column headers indicating presence in Pemberton et al.  or Kyriazopoulou-Panagiotopoulou et al. . We provide the estimated relationship coefficient for pairs that we were able to reconstruct according to the methods. This list includes all relationships with a K1 greater than 0.025 (including ID/MZ that have K2 ∼1.0) as well as the relationships between the parents of inbred individuals.
[Show abstract][Hide abstract] ABSTRACT: Evidence for IBD2 sharing between individuals. For four pairs of related individuals who were annotated as related (either previously or in this study), we show IBD2 sharing across various chromosomes as provided by SNPduo analysis. For each pairwise comparison the three tracks are IBS0, IBS1, and IBS2. (A) Previously annotated second-degree relationship between LWK members NA19334 and NA19313 has unexpected IBD2 sharing on chromosome 19. (B) Previously annotated second-degree relationship (inferred by us to be avuncular) between MKK members NA21362 and NA21438 has IBD2 sharing across large regions of chromosome 1. Note that this pair had 10/15 full-sibling annotations given by RELPAIR from Pemberton et al . (C) Newly annotated relationship of an unknown degree between MXL members NA19657 (mother of family M007) and NA19787 (son of family M032) has IBD2 sharing on chromosome 9. (D) Previously annotated avuncular relationship between LWK members NA19443 and NA19469 has IBD2 sharing on chromosome 4.
[Show abstract][Hide abstract] ABSTRACT: It is an assumption of large, population-based datasets that samples are annotated accurately whether they correspond to known relationships or unrelated individuals. These annotations are key for a broad range of genetics applications. While many methods are available to assess relatedness that involve estimates of identity-by-descent (IBD) and/or identity-by-state (IBS) allele-sharing proportions, we developed a novel approach that estimates IBD0, 1, and 2 based on observed IBS within windows. When combined with genome-wide IBS information, it provides an intuitive and practical graphical approach with the capacity to analyze datasets with thousands of samples without prior information about relatedness between individuals or haplotypes. We applied the method to a commonly used Human Variation Panel consisting of 400 nominally unrelated individuals. Surprisingly, we identified identical, parent-child, and full-sibling relationships and reconstructed pedigrees. In two instances non-sibling pairs of individuals in these pedigrees had unexpected IBD2 levels, as well as multiple regions of homozygosity, implying inbreeding. This combined method allowed us to distinguish related individuals from those having atypical heterozygosity rates and determine which individuals were outliers with respect to their designated population. Additionally, it becomes increasingly difficult to identify distant relatedness using genome-wide IBS methods alone. However, our IBD method further identified distant relatedness between individuals within populations, supported by the presence of megabase-scale regions lacking IBS0 across individual chromosomes. We benchmarked our approach against the hidden Markov model of a leading software package (PLINK), showing improved calling of distantly related individuals, and we validated it using a known pedigree from a clinical study. The application of this approach could improve genome-wide association, linkage, heterozygosity, and other population genomics studies that rely on SNP genotype data.
[Show abstract][Hide abstract] ABSTRACT: Partial monosomy 21 was recently segregated into three regions associated with variable clinical severity. We describe 10 new patients, all examined by single nucleotide polymorphism (SNP) genotyping and G-banded karyotyping. Cohort A consisted of three patients seen in our medical genetics clinics with partial chromosome 21 monosomies. In two of these patients having terminal deletions (21q22.2-ter and 21q22.3-ter), the breakpoints differed by at least 812 Kb of sequence, containing seven RefSeq genes. A third patient had an interstitial hemizygous loss of 16.4 Mb (21q21.1-q22.11). All three patients had relatively mild phenotypes. Cohort B consisted of seven patients with partial chromosome 21 monosomies who had a greater number of dysmorphic features and some major malformations; SNP genotypes were obtained from the Coriell Genetic Cell Repository. We also collected data on partial monsomy 21 cases from the DECIPHER database. This report of 10 new cases of 21q deletion and review of a total of 36 confirms that deletion of the terminal region is associated with a mild phenotype, but suggests that deletion of regions 1 and 2 is compatible with life and have a variable phenotype perhaps relating more to other genetic and environmental variables than to genes in the interval.
Full-text · Article · Feb 2011 · European journal of human genetics: EJHG
[Show abstract][Hide abstract] ABSTRACT: Metastasis is a defining feature of malignant tumors and is the most common cause of cancer-related death, yet the genetics
of metastasis are poorly understood. We used exome capture coupled with massively parallel sequencing to search for metastasis-related
mutations in highly metastatic uveal melanomas of the eye. Inactivating somatic mutations were identified in the gene encoding
BRCA1-associated protein 1 (BAP1) on chromosome 3p21.1 in 26 of 31 (84%) metastasizing tumors, including 15 mutations causing premature protein termination
and 5 affecting its ubiquitin carboxyl-terminal hydrolase domain. One tumor harbored a frameshift mutation that was germline
in origin, thus representing a susceptibility allele. These findings implicate loss of BAP1 in uveal melanoma metastasis and
suggest that the BAP1 pathway may be a valuable therapeutic target.
[Show abstract][Hide abstract] ABSTRACT: Psoriasis is a common incurable inflammatory skin disease affecting 2-3% of the European population. Psoriatic skin contains large numbers of immune cells which produce many cytokines, chemokines and inflammatory molecules. The epidermis divides much faster than normal and has a defective outer layer or barrier which under normal circumstances protects from infection and dehydration. Psoriatic skin is characterized by a distinct set of inflammation and epidermal proliferation and differentiation markers, and it has been unclear whether the genetic basis of psoriasis reflects defects of the immune system or of the skin. One genetic determinant lies within the major histocompatibility complex class 1 region. Genome-wide association studies have revealed genetic susceptibility factors that play a role in the formation of immune cells found in psoriasis lesions. Others affect epidermal proliferation and skin barrier formation. Hence, genetic components of both the immune system and the epidermis can predispose to disease.
[Show abstract][Hide abstract] ABSTRACT: Meiotic crossovers are the major mechanism by which haplotypes are shuffled to generate genetic diversity. Previously available methods for the genome-wide, high-resolution identification of meiotic crossover sites are limited by the laborious nature of the assay (as in sperm typing).
Several methods have been introduced to identify crossovers using high density single nucleotide polymorphism (SNP) array technologies, although programs are not widely available to implement such analyses.
Here we present a two-generation "reverse pedigree analysis" method (analyzing the genotypes of two children relative to each parent) and a web-accessible tool to determine and visualize inheritance differences among siblings and crossover locations on each parental gamete. This approach is complementary to existing methods and uses informative markers which provide high resolution for locating meiotic crossover sites. We introduce a segmentation algorithm to identify crossover sites, and used a synthetic data set to determine that the segmentation algorithm specificity was 92% and sensitivity was 89%. The use of reverse pedigrees allows the inference of crossover locations on the X chromosome in a maternal gamete through analysis of two sons and their father. We further analyzed genotypes from eight multiplex autism families, observing a 1.462 maternal to paternal recombination ratio and no significant differences between affected and unaffected children. Meiotic recombination results from pediSNP can also be used to identify haplotypes that are shared by probands within a pedigree, as we demonstrated with a multiplex autism family.
Using "reverse pedigrees" and defining unique sets of genotype markers within pedigree data, we introduce a method that identifies inherited allelic differences and meiotic crossovers. We implemented the method in the pediSNP software program, and we applied it to several data sets. This approach uses data from two generations to identify crossover sites, facilitating studies of recombination in disease. pediSNP is available online at http://pevsnerlab.kennedykrieger.org/pediSNP.
Full-text · Article · Sep 2009 · BMC Medical Genetics