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The major determinant of disease severity in Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD) is whether the dystrophin gene (DMD) mutation truncates the mRNA reading frame or allows expression of a partially functional protein. However, even in the complete absence of dystrophin, variability in disease severity is observed, and candidate gene studies have implicated several genes as modifiers. Here we present the largest genome-wide search to date for loci influencing severity in N= 419 DMD patients. Availability of subjects for such studies is quite limited, leading to modest sample sizes, which present a challenge for GWAS design. We have therefore taken special steps to minimize heterogeneity within our dataset at the DMDlocus itself, taking a novel approach to mutation classification to effectively exclude the possibility of residual dystrophin expression, and utilized statistical methods that are well adapted to smaller sample sizes, including the use of a novel linear regression-like residual for time to ambulatory loss and the application of evidential statistics for the GWAS approach. Finally, we applied an unbiased in silico pipeline, utilizing functional genomic datasets to explore the potential impact of the best supported SNPs. In all, we obtained eight SNPs (out of 1,385,356 total) with posterior probability of trait-marker association (PPLD) ≥ 0.4, representing six distinct loci. Our analysis prioritized likely non-coding SNP regulatory effects on six genes (ETAA1, PARD6G, GALNTL6, MAN1A1, ADAMTS19, and NCALD), each with plausibility as a DMD modifier. These results support both recurrent and potentially new pathways for intervention in the dystrophinopathies.

Fascioscapulohumeral muscular dystrophy (FSHD) is caused by a unique genetic mechanism that relies on contraction and hypomethylation of the D4Z4 macrosatellite array on the Chromosome 4q telomere allowing ectopic expression of the DUX4gene in skeletal muscle. Genetic analysis is difficult because of the large size and repetitive nature of the array, a nearly identical array on the 10q telomere, and the presence of divergent D4Z4 arrays scattered throughout the genome. Here, we combine nanopore long-read sequencing with Cas9-targeted enrichment of 4q and 10q D4Z4 arrays for comprehensive genetic analysis including determination of the length of the 4q and 10q D4Z4 arrays with base-pair resolution. In the same assay, we differentiate 4q from 10q telomeric sequences, determine A/B haplotype, identify paralogous D4Z4 sequences elsewhere in the genome, and estimate methylation for all CpGs in the array. Asymmetric, length-dependent methylation gradients were observed in the 4q and 10q D4Z4 arrays that reach a hypermethylation point at approximately 10 D4Z4 repeat units, consistent with the known threshold of pathogenic D4Z4 contractions. High resolution analysis of individual D4Z4 repeat methylation revealed areas of low methylation near the CTCF/insulator region and areas of high methylation immediately preceding the DUX4transcriptional start site. Within the DUX4exons, we observed a waxing/waning methylation pattern with a 180-nucleotide periodicity, consistent with phased nucleosomes. Targeted nanopore sequencing complements recently developed molecular combing and optical mapping approaches to genetic analysis for FSHD by adding precision of the length measurement, base-pair resolution sequencing, and quantitative methylation analysis.

The observation that Borrelia burgdorferi-induced arthritis is severe in C3H mice and milder in C57BL/6 (B6) mice has allowed a forward genetics approach for the identification of genetic elements that regulate the arthritis response. Quantitative trait loci (QTL) on five chromosomes (Chr) were identified previously in segregating crosses between C3H and B6 mice and collectively designated B. burgdorferi arthritis-associated (Bbaa) QTL. Reciprocal interval-specific congenic lines (ISCL) that encompass Bbaa1, Bbaa2-Bbaa3, Bbaa4, Bbaa6, and Bbaa12 on Chr 4, 5, 11, 12, and 1, respectively, have now been generated. Bidirectional transfer of the arthritis severity phenotype in association with Bbaa2-Bbaa3 and Bbaa4 was observed, and unidirectional transfer with the B6 allele of Bbaa6 was noted. These findings confirm the existence of polymorphic loci within Bbaa2-Bbaa3, Bbaa4, and Bbaa6 that regulate the severity of B. burgdorferi-induced arthritis. ISCL were used to assess the regulation of a previously identified interferon transcriptional profile associated with severe disease in C3H mice. The regulation of this transcriptional signature was found to be independent of penetrant Bbaa QTL, both in joint tissues and in isolated macrophages. These results clearly demonstrate the utility of forward genetics for the discovery of novel genes and pathways involved in the regulation of the severity of Lyme arthritis and predict the involvement of regulatory elements not evident from other experimental approaches.

ABSTRACTThe observation that Borrelia burgdorferi-induced arthritis is severe in C3H mice and milder in C57BL/6 (B6) mice has allowed a forward genetics approach for the identification of genetic elements that regulate the arthritis response. Quantitative trait loci (QTL) on five chromosomes (Chr) were identified previously in segregating crosses between C3H and B6 mice and collectively designated B. burgdorferiarthritis-associated (Bbaa) QTL. Reciprocal interval-specific congenic lines (ISCL) that encompass Bbaa1, Bbaa2-Bbaa3, Bbaa4, Bbaa6, and Bbaa12on Chr 4, 5, 11, 12, and 1, respectively, have now been generated. Bidirectional transfer of the arthritis severity phenotype in association with Bbaa2-Bbaa3and Bbaa4was observed, and unidirectional transfer with the B6 allele of Bbaa6was noted. These findings confirm the existence of polymorphic loci within Bbaa2-Bbaa3, Bbaa4, and Bbaa6that regulate the severity of B. burgdorferi-induced arthritis. ISCL were used to assess the regulation of a previously identified interferon transcriptional profile associated with severe disease in C3H mice. The regulation of this transcriptional signature was found to be independent of penetrant BbaaQTL, both in joint tissues and in isolated macrophages. These results clearly demonstrate the utility of forward genetics for the discovery of novel genes and pathways involved in the regulation of the severity of Lyme arthritis and predict the involvement of regulatory elements not evident from other experimental approaches.

Hepatic siderosis is common in patients with porphyria cutanea tarda (PCT). Mutations in the hereditary hemochromatosis (hh) gene (HFE) explain the siderosis in approximately 20% patients, suggesting that the remaining occurrences result from additional genetic and environmental factors. Two genes known to modify iron loading in hh are hepcidin (HAMP) and hemojuvelin (HJV). To determine if mutations in or expression of these genes influenced iron overload in PCT, we compared sequences of HAMP and HJV in 96 patients with PCT and 88 HFE C282Y homozygotes with marked hepatic iron overload. We also compared hepatic expression of these and other iron-related genes in a group of patients with PCT and hh. Two intronic polymorphisms in HJV were associated with elevated serum ferritin in HFE C282Y homozygotes. No exonic polymorphisms were identified. Sequencing of HAMP revealed exonic polymorphisms in 2 patients with PCT: heterozygosity for a G→A transition (G71D substitution) in one and heterozygosity for an A→G transition (K83R substitution) in the other. Hepatic HAMP expression in patients with PCT was significantly reduced, regardless of HFE genotype, when compared with patients with hh but without PCT with comparable iron overload. These data indicate that the hepatic siderosis associated with PCT likely results from dysregulated HAMP.

Hepatic siderosis is common in patients with porphyria cutanea tarda (PCT). Mutations in the hereditary hemochromatosis (hh) gene (HFE) explain the siderosis in approximately 20% patients, suggesting that the remaining occurrences result from additional genetic and environmental factors. Two genes known to modify iron loading in hh are hepcidin (HAMP) and hemojuvelin (HJV). To determine if mutations in or expression of these genes influenced iron overload in PCT, we compared sequences of HAMPand HJVin 96 patients with PCT and 88 HFEC282Y homozygotes with marked hepatic iron overload. We also compared hepatic expression of these and other iron-related genes in a group of patients with PCT and hh. Two intronic polymorphisms in HJVwere associated with elevated serum ferritin in HFEC282Y homozygotes. No exonic polymorphisms were identified. Sequencing of HAMPrevealed exonic polymorphisms in 2 patients with PCT: heterozygosity for a G→A transition (G71D substitution) in one and heterozygosity for an A→G transition (K83R substitution) in the other. Hepatic HAMPexpression in patients with PCT was significantly reduced, regardless of HFEgenotype, when compared with patients with hh but without PCT with comparable iron overload. These data indicate that the hepatic siderosis associated with PCT likely results from dysregulated HAMP.

Gene expression profiling of early eosinophil development shows increased transcript levels of proinflammatory cytokines, chemokines, transcription factors, and a novel gene, EGO(eosinophil granule ontogeny). EGOis nested within an intron of the inositol triphosphate receptor type 1 (ITPR1)gene and is conserved at the nucleotide level; however, the largest open reading frame (ORF) is 86 amino acids. Sucrose density gradients show that EGOis not associated with ribosomes and therefore is a noncoding RNA (ncRNA). EGOtranscript levels rapidly increase following interleukin-5 (IL-5) stimulation of CD34+hematopoietic progenitors. EGORNA also is highly expressed in human bone marrow and in mature eosinophils. RNA silencing of EGOresults in decreased major basic protein (MBP) and eosinophil derived neurotoxin (EDN) mRNA expression in developing CD34+hematopoietic progenitors in vitro and in a CD34+cell line model. Therefore, EGOis a novel ncRNA gene expressed during eosinophil development and is necessary for normal MBPand EDNtranscript expression.

Gene expression profiling of early eosinophil development shows increased transcript levels of proinflammatory cytokines, chemokines, transcription factors, and a novel gene, EGO (eosinophil granule ontogeny). EGO is nested within an intron of the inositol triphosphate receptor type 1 (ITPR1) gene and is conserved at the nucleotide level; however, the largest open reading frame (ORF) is 86 amino acids. Sucrose density gradients show that EGO is not associated with ribosomes and therefore is a noncoding RNA (ncRNA). EGO transcript levels rapidly increase following interleukin-5 (IL-5) stimulation of CD34+ hematopoietic progenitors. EGO RNA also is highly expressed in human bone marrow and in mature eosinophils. RNA silencing of EGO results in decreased major basic protein (MBP) and eosinophil derived neurotoxin (EDN) mRNA expression in developing CD34+ hematopoietic progenitors in vitro and in a CD34+ cell line model. Therefore, EGO is a novel ncRNA gene expressed during eosinophil development and is necessary for normal MBP and EDN transcript expression.

Several quantitative trait loci regulating murine Lyme arthritis severity have been mapped, including a highly significant linkage found on chromosome 5, termed Bb2Bb3. Within this region, the Ncf1gene of the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been identified as a major regulator of arthritis severity in rodent models of rheumatoid arthritis, an effect attributed to protective properties of reactive oxygen species. To assess the role of Ncf1in Lyme arthritis, we introgressed Bb2Bb3from severely arthritic C3H/He mice onto mildly arthritic C57BL/6 mice. This increased Lyme arthritis severity, whereas the reciprocal transfer conferred protection from disease. A single nucleotide polymorphism was identified in the Ncf1gene that did not influence the protein sequence or expression of Ncf1. Although polymorphonuclear leukocytes from C57BL/6 mice generated a greater oxidative burst than polymorphonuclear leukocytes from C3H/He mice, studies with the Bb2Bb3congenic mice demonstrated this difference was not linked to Ncf1alleles. Furthermore, Lyme arthritis severity was not altered in mice lacking either the Ncf1or Gp91phoxsubunits of the NADPH oxidase complex. Together, these results argue that Ncf1is not a candidate gene for regulation of Lyme arthritis and reveal Lyme arthritis to be independent of NADPH oxidase activity, distinguishing it from other models of rheumatoid arthritis.

Initiatives to sequence DNA on a large scale have created a need for increased throughput and decreased costs. One scheme for increasing throughput, multiplex sequencing, involves the processing of a mixture of sequencing templates followed by sequential hybridization to reveal the individual sequence ladders on a membrane. Because multiplex sequencing has not been fully automated, and has not seemed automatable, few sequencing efforts have attempted to exploit it. We describe here a scheme for the automation of multiplex sequencing. Probe hybridized to target DNA is detected via spatially localized enzyme-linked fluorescence. Light output is high enough that imaging is possible with simple instrumentation. Direct imaging within an automated hybridization apparatus is made feasible so that the entire process will be automatic once a multiplex membrane is produced. The technique has the potential to increase severalfold the throughput of automated sequencing instruments required for sequencing the human genome. Copyright 1994, 1999 Academic Press

Homozygosity for the HFE C282Y mutation accounts for approximately 90 percent of HFE-associated (type 1) hereditary hemochromatosis. The clinical phenotype in C282Y homozygotes, however, ranges from simply an elevated percent saturation of transferrin to organ damage due to iron overload. Modifier genes have been proposed to explain this phenotypic variability. Hepatic siderosis is a nearly constant finding in patients with PCT. Approximately 20 percent of patients with PCT are homozygotes for the C282Y HFE mutation but the cause of hepatic iron loading in the remaining 80 percent is not known. Approximately one-third of patients with PCT are heterozygotes for mutations of the uroporphyrinogen decarboxylase (URO-D) gene (familial PCT) but pedigree studies indicate that clinical expression occurs only in those URO-D heterozygotes who develop hepatic siderosis. Most patients with PCT have no URO-D mutations (sporadic PCT) but virtually all sporadic cases have hepatic iron overload. Two genes known to affect iron homeostasis are hepcidin (HAMP) and hemojuvelin (HJV). Heterozygosity for HAMP and HJV mutations have been associated with marked iron overload in a small number of patients with type 1 hemochromatosis (Blood.2004; 103:2835–40; Blood Cells Mol Dis.2004; 33:338–43). We asked if mutations of HAMP or HJV could account for hepatic iron overload in highly penetrant C282Y homozygotes and in PCT patients with or without HFE mutations. We sequenced the HAMP and HJV genes in 96 hemochromatosis patients with grade 3–4 (scale 0–4) hepatic parenchymal cell stainable iron (HPCSI) and 96 PCT patients with variable degrees of hepatic siderosis. Ninety-four percent (90) of the hemochromatosis patients were C282Y homozygotes, 4.2 percent (4) were C282Y heterozygotes and 2.1 percent (2) were wild type 282 homozygotes. No exonic changes or splice site mutations were detected in either the HAMP or HJV genes. Eighty-three of the 96 PCT patients were genotyped at the HFE locus. Twenty-five percent (21) were C282Y homozygotes, 23 percent (19) were C282Y heterozygotes and 52 percent (43) were wild type 282 homozygotes. No exonic changes or splice site mutations were detected in the HJV gene of patients with PCT but two PCT patients were found to be heterozygotes for HAMP mutations. The first had the previously identified 212G?A transition leading to a G71D substitution. The second had a 248A?C transversion corresponding to K83R in the peptide. Both of these PCT patients were HFE 282 wild type homozygotes but both had grade 4 HPCSI. These data indicate that heterozygosity for mutations of HAMP or HJV rarely modifies the iron loading phenotype in either type 1 hemochromatosis or PCT. Other modifier loci must exist.

Homozygosity for the HFE C282Y mutation accounts for approximately 90 percent of HFE-associated (type 1) hereditary hemochromatosis. The clinical phenotype in C282Y homozygotes, however, ranges from simply an elevated percent saturation of transferrin to organ damage due to iron overload. Modifier genes have been proposed to explain this phenotypic variability. Hepatic siderosis is a nearly constant finding in patients with PCT. Approximately 20 percent of patients with PCT are homozygotes for the C282Y HFE mutation but the cause of hepatic iron loading in the remaining 80 percent is not known. Approximately one-third of patients with PCT are heterozygotes for mutations of the uroporphyrinogen decarboxylase (URO-D) gene (familial PCT) but pedigree studies indicate that clinical expression occurs only in those URO-D heterozygotes who develop hepatic siderosis. Most patients with PCT have no URO-D mutations (sporadic PCT) but virtually all sporadic cases have hepatic iron overload. Two genes known to affect iron homeostasis are hepcidin (HAMP) and hemojuvelin (HJV). Heterozygosity for HAMP and HJV mutations have been associated with marked iron overload in a small number of patients with type 1 hemochromatosis (Blood. 2004; 103:2835–40; Blood Cells Mol Dis. 2004; 33:338–43). We asked if mutations of HAMP or HJV could account for hepatic iron overload in highly penetrant C282Y homozygotes and in PCT patients with or without HFE mutations. We sequenced the HAMP and HJV genes in 96 hemochromatosis patients with grade 3–4 (scale 0–4) hepatic parenchymal cell stainable iron (HPCSI) and 96 PCT patients with variable degrees of hepatic siderosis. Ninety-four percent (90) of the hemochromatosis patients were C282Y homozygotes, 4.2 percent (4) were C282Y heterozygotes and 2.1 percent (2) were wild type 282 homozygotes. No exonic changes or splice site mutations were detected in either the HAMP or HJV genes. Eighty-three of the 96 PCT patients were genotyped at the HFE locus. Twenty-five percent (21) were C282Y homozygotes, 23 percent (19) were C282Y heterozygotes and 52 percent (43) were wild type 282 homozygotes. No exonic changes or splice site mutations were detected in the HJV gene of patients with PCT but two PCT patients were found to be heterozygotes for HAMP mutations. The first had the previously identified 212G→A transition leading to a G71D substitution. The second had a 248A→C transversion corresponding to K83R in the peptide. Both of these PCT patients were HFE 282 wild type homozygotes but both had grade 4 HPCSI. These data indicate that heterozygosity for mutations of HAMP or HJV rarely modifies the iron loading phenotype in either type 1 hemochromatosis or PCT. Other modifier loci must exist.