Emebet Mengesha

Missense Mutation in Pseudouridine Synthase 1 (PUS1) Causes Mitochondrial Myopathy and Sideroblastic Anemia (MLASA)

Mitochondrial myopathy and sideroblastic anemia (MLASA) is a rare, autosomal recessive oxidative phosphorylation disorder specific to skeletal muscle and bone marrow. Linkage analysis and homozygosity testing of two families with MLASA localized the candidate region to 1.2 Mb on 12q24.33. Sequence analysis of each of the six known genes in this region, as well as four putative genes with expression in bone marrow or muscle, identified a homozygous missense mutation in the pseudouridine synthase 1 gene (PUS1) in all patients with MLASA from these families. The mutation is the only amino acid coding change in these 10 genes that is not a known polymorphism, and it is not found in 934 controls. The amino acid change affects a highly conserved amino acid, and appears to be in the catalytic center of the protein, PUS1p. PUS1 is widely expressed, and quantitative expression analysis of RNAs from liver, brain, heart, bone marrow, and skeletal muscle showed elevated levels of expression in skeletal muscle and brain. We propose deficient pseudouridylation of mitochondrial tRNAs as an etiology of MLASA. Identification of the pathophysiologic pathways of the mutation in these families may shed light on the tissue specificity of oxidative phosphorylation disorders.

Mutation in TRMU Related to Transfer RNA Modification Modulates the Phenotypic Expression of the Deafness-Associated Mitochondrial 12S Ribosomal RNA Mutations

The human mitochondrial 12S ribosomal RNA (rRNA) A1555G mutation has been associated with aminoglycoside-induced and nonsyndromic deafness in many families worldwide. Our previous investigation revealed that the A1555G mutation is a primary factor underlying the development of deafness but is not sufficient to produce a deafness phenotype. However, it has been proposed that nuclear-modifier genes modulate the phenotypic manifestation of the A1555G mutation. Here, we identified the nuclear-modifier gene TRMU, which encodes a highly conserved mitochondrial protein related to transfer RNA (tRNA) modification. Genotyping analysis of TRMU in 613 subjects from 1 Arab-Israeli kindred, 210 European (Italian pedigrees and Spanish pedigrees) families, and 31 Chinese pedigrees carrying the A1555G or the C1494T mutation revealed a missense mutation (G28T) altering an invariant amino acid residue (A10S) in the evolutionarily conserved N-terminal region of the TRMU protein. Interestingly, all 18 Arab-Israeli/Italian-Spanish matrilineal relatives carrying both the TRMU A10S and 12S rRNA A1555G mutations exhibited prelingual profound deafness. Functional analysis showed that this mutation did not affect importation of TRMU precursors into mitochondria. However, the homozygous A10S mutation leads to a marked failure in mitochondrial tRNA metabolisms, specifically reducing the steady-state levels of mitochondrial tRNA. As a consequence, these defects contribute to the impairment of mitochondrial-protein synthesis. Resultant biochemical defects aggravate the mitochondrial dysfunction associated with the A1555G mutation, exceeding the threshold for expressing the deafness phenotype. These findings indicate that the mutated TRMU, acting as a modifier factor, modulates the phenotypic manifestation of the deafness-associated 12S rRNA mutations.

IL-23 Receptor (IL-23R) Gene Protects Against Pediatric Crohn’s Disease

Background The IL‐23 receptor (IL‐23R) has been found to be associated with small bowel Crohns disease (CD) in a whole genome association study. Specifically, the rare allele of the R381Q single nucleotide polymorphism (SNP) conferred protection against CD. It is unknown whether IL‐23R is associated with IBD in children. The aim was to examine the association of IL‐23R with susceptibility to IBD in pediatric patients. Methods DNA was collected from 609 subjects (151 CD and 52 ulcerative colitis [UC] trios). Trios were genotyped for the R381Q SNP of the IL‐23R gene and SNP8, SNP12, SNP13, of the CARD15 gene using Taqman. The transmission disequilibrium test (TDT) was used for association to disease using GENEHUNTER 2.0. Results The rare allele of R381Q SNP was present in 2.7% of CD and 2.9% UC probands. The CARD15 frequency was 31.5% (CD) and 18% (UC). The IL‐23R allele was negatively associated with inflammatory bowel disease (IBD): the R381Q SNP was undertransmitted in children with IBD (8 transmitted [T] versus 27 untransmitted [UT]; P = 0.001). This association was significant for all CD patients (6 T versus 19 UT; P = 0.009), especially for non‐Jewish CD patients (2 T versus 17 UT; P = 0.0006). TDT showed a borderline association for UC (2 T versus 8 UT; P = 0.06). As expected, CARD15 was associated with CD in children by the TDT (58 T versus 22 UT P = 0.00006), but not with UC. Conclusions The protective IL‐23R R381Q variant was particularly associated with CD in non‐Jewish children. Thus, the initial whole genome association study based on ileal CD in adults has been extended to the pediatric population and beyond small bowel CD. (Inflamm Bowel Dis 2007)

Genetic Predictors of Medically Refractory Ulcerative Colitis

Background: Acute severe ulcerative colitis (UC) remains a significant clinical challenge and the ability to predict, at an early stage, those individuals at risk of colectomy for medically refractory UC (MR‐UC) would be a major clinical advance. The aim of this study was to use a genome‐wide association study (GWAS) in a well‐characterized cohort of UC patients to identify genetic variation that contributes to MR‐UC. Methods: A GWAS comparing 324 MR‐UC patients with 537 non‐MR‐UC patients was analyzed using logistic regression and Cox proportional hazards methods. In addition, the MR‐UC patients were compared with 2601 healthy controls. Results: MR‐UC was associated with more extensive disease (P = 2.7 × 10−6) and a positive family history of UC (P = 0.004). A risk score based on the combination of 46 single nucleotide polymorphisms (SNPs) associated with MR‐UC explained 48% of the variance for colectomy risk in our cohort. Risk scores divided into quarters showed the risk of colectomy to be 0%, 17%, 74%, and 100% in the four groups. Comparison of the MR‐UC subjects with healthy controls confirmed the contribution of the major histocompatibility complex to severe UC (peak association: rs17207986, P = 1.4 × 10−16) and provided genome‐wide suggestive association at the TNFSF15 (TL1A) locus (peak association: rs11554257, P = 1.4 × 10−6). Conclusions: A SNP‐based risk scoring system, identified here by GWAS analyses, may provide a useful adjunct to clinical parameters for predicting the natural history of UC. Furthermore, discovery of genetic processes underlying disease severity may help to identify pathways for novel therapeutic intervention in severe UC. (Inflamm Bowel Dis 2010)

Genetic epistasis of IL23/IL17 pathway genes in Crohn's disease

Background: The IL23/IL17 pathway is pivotal in the development of chronic mucosal inflammation seen in Crohns disease (CD). Genetic variants in the IL23R and IL12B have been associated with CD susceptibility. We investigated 10 genes within the IL23/IL17 pathway in a case‐control study of 763 CD cases and 254 healthy controls. Methods: We identified a novel association in haplotypes in IL17A (empirical P = 0.02), IL17RA (P = 0.001), IL17RD (P = 0.001), IL12RB1 (P = 0.003), and IL12RB2 (P = 0.001) as well as confirming the association with IL12B variants (P = 0.003). Results: The cumulative risk for carrying an increased number of CD risk haplotypes from genes in this pathway rises to an odds ratio of 4.3 for carrying 5 risk haplotypes. We have previously demonstrated an association between this cohort and IL23R haplotypes. Pairwise analyses suggest that there is statistical interaction between variants in IL17A and IL23R (P = 0.047) and between variants in IL17RA and IL23R (P = 0.036). Furthermore, a significant association between CD and the widely replicated IL23R variants is only seen in the presence of IL17A or IL17RA variants. Conclusions: These data support the investigation of pathways implicated in CD pathogenesis in order to identify further susceptibility genes and also suggest that important gene–gene interaction is present in CD susceptibility.

IL23R Haplotypes Provide a Large Population Attributable Risk for Crohn's Disease

Background: The IL‐23 pathway plays a pivotal role in the development of chronic mucosal inflammation seen in the inflammatory bowel diseases. Multiple studies have now established the contribution of the interleukin 23 receptor gene (IL23R) to Crohns disease (CD) risk in general and of the IL23R R381Q variant in particular. The aim of this work was to estimate the total contribution of this gene to CD risk test using a haplotype approach. Methods: In all, 763 CD subjects and 254 controls were genotyped for single nucleotide polymorphisms in the IL23R gene using Illumina and ABI methods. Haplotypes were assigned using PHASEv2 and tested for association with CD by chi‐square and permutation. Results: Haplotypes with both increased and decreased risk for CD were observed in 2 of the 4 observed blocks (Block 2 H1: 55.4% control, 64% CD, P = 0.019; H2: 64.5% control, 54.4% CD, P = 0.006; Block 3 H1: 55.8% control, 64.4% CD, P = 0.013; H2: 47.0% control, 36.6% CD, P = 0.001). The population attributable risk for these haplotypes was substantially larger than that estimated for the IL23R R381Q variant (Block 2 H1 and block 3 H1 ≈20%, compared with ≈4% for Block 3 H6, containing the variant). Conclusions: These observations suggest that IL23R makes a substantial contribution to CD susceptibility, larger than that estimated from the population frequency of the R381Q variant. These observations also support the expectation that finding “hits” from genomewide association studies will be but an important chapter in the story of unraveling the genetic contribution to CD, rather than the final chapter that brings clarity to all the plot twists of a complicated story.

MAGI2 genetic variation and inflammatory bowel disease.

Background: Despite recent advances the majority of inflammatory bowel disease (IBD) susceptibility ‘genes’ remain undiscovered. Recent data suggest that autoimmune conditions may ‘share’ susceptibility loci. Epidemiological evidence indicates an association between celiac disease and IBD and both conditions demonstrate increased gut permeability. MAGI2, recently implicated in ulcerative colitis (UC) and celiac disease, encodes a scaffolding protein involved in epithelial integrity. Our aim was to test MAGI2 variants for association with IBD and also their role in determining intermediate hereditary phenotypes defined by antibody production to microbial antigens. Methods: We genotyped 113 MAGI2 single nucleotide polymorphisms (SNPs) in 681 cases of Crohns disease (CD), 259 UC cases, and 195 controls. Results: The most significant IBD association was in intron 6 (rs2160322, P = 0.009) and both UC (P = 0.006) and CD (P = 0.03) contributed to this association. The most significant CD association was with an intron 2 haplotype (rs7785088/rs323149/rs13246026, P = 0.002). We observed highly significant associations with UC in intron 6 (rs7803276/rs7803705, P = 0.002) and also significant associations in introns 2, 6, and 20. Significant associations were seen with: immunoglobulin G (IgG) anti‐Saccharomyces cerevisiae antibodies (ASCA)‐positive CD in intron 3 (P = 0.003), intron 6 (P = 0.003), and intron 20 (P = 0.001); anti‐CBir1‐positive CD in intron 3 (P = 0.0001) and intron 6 (P = 0.008); and anti‐outer membrane porin C (OmpC)‐positive CD in intron 3 (P = 0.0009), and intron 9 (P = 0.007). Quantitative antibody levels were also associated with variants in intron 4 (anti‐IgA ASCA, P = 0.0003 and anti‐IgG ASCA, P = 0.0002). Conclusions: These findings support the significance of the epithelial barrier in IBD pathogenesis.

Systems Genetics Reveals the Functional Context of PCOS Loci and Identifies Genetic and Molecular Mechanisms of Disease Heterogeneity

Genome wide association studies (GWAS) have revealed 11 independent risk loci for polycystic ovary syndrome (PCOS), a common disorder in young women characterized by androgen excess and oligomenorrhea. To put these risk loci and the single nucleotide polymorphisms (SNPs) therein into functional context, we measured DNA methylation and gene expression in subcutaneous adipose tissue biopsies to identify PCOS-specific alterations. Two genes from the LHCGR region, STON1-GTF2A1L and LHCGR, were overexpressed in PCOS. In analysis stratified by obesity, LHCGR was overexpressed only in non-obese PCOS women. Although not differentially expressed in the entire PCOS group, INSR was underexpressed in obese PCOS subjects only. Alterations in gene expression in the LHCGR, RAB5B and INSR regions suggest that SNPs in these loci may be functional and could affect gene expression directly or indirectly via epigenetic alterations. We identified reduced methylation in the LHCGR locus and increased methylation in the INSR locus, changes that are concordant with the altered gene expression profiles. Complex patterns of meQTL and eQTL were identified in these loci, suggesting that local genetic variation plays an important role in gene regulation. We propose that non-obese PCOS women possess significant alterations in LH receptor expression, which drives excess androgen secretion from the ovary. Alternatively, obese women with PCOS possess alterations in insulin receptor expression, with underexpression in metabolic tissues and overexpression in the ovary, resulting in peripheral insulin resistance and excess ovarian androgen production. These studies provide a genetic and molecular basis for the reported clinical heterogeneity of PCOS.

Variants in ZNF365 isoform D are associated with Crohn's disease

Objective Genome-wide association studies have identified multiple Crohns disease (CD) susceptibility loci, including association with non-coding intergenic single-nucleotide polymorphisms (SNPs) at 10q21. Design To fine-map the 10q21 locus, the authors genotyped 86 SNPs in 1632 CD cases and 961 controls and performed single-marker and conditional analyses using logistic regression. Results Association with CD risk spanning 11 SNPs (p<0.001) was observed. The most significant association observed was at the non-synonymous SNP, rs7076156 (Ala62Thr), in ZNF365. The alanine allele was over-represented in CD (p=5.23×10−7; OR=1.39 (95% CI 1.22 to 1.58)); allele frequency of 76% in CD and 69.7% in controls). Conditional analysis on rs7076156 nullified all other significant associations, suggesting that this is the causative variant at this locus. Four isoforms of ZNF365 have previously been identified, and rs7076156 is located in an exon unique to ZNF365 isoform D. The authors demonstrated, using reverse transcription-PCR, expression of ZNF365D in intestinal resections from both CD subjects and controls. Markedly reduced mean expression levels of ZNF365D were identified in Epstein–Barr virus-transformed lymphoblastoid cell lines from CD subjects homozygous for the risk allele (Ala). A whole-genome microarray expression study further suggested that the Ala62Thr change in ZNF365 isoform D is related to differential expression of the genes ARL4A, MKKS, RRAGD, SUMF2, TDR1 and ZNF148 in CD. Conclusions Collectively, these data support the hypothesis that the non-synonymous Ala62Thr SNP, rs7076156, underlies the association between 10q21 and CD risk and suggest that this SNP acts by altering expression of genes under the control of ZNF365 isoform D.

Gene responsible for mitochondrial myopathy and sideroblastic anemia (MSA) maps to chromosome 12q24.33

Mitochondrial myopathy and sideroblastic anemia (MSA) is a rare autosomal recessive disorder of oxidative phosphorylation and iron metabolism. Individuals with MSA present with weakness and anemia in late childhood and may become dependent on blood transfusions. Recently, we reported affected sibling pairs from a Jewish‐Iranian kindred living in the US [Casas and Fischel‐Ghodsian, 2003]. A genome scan and fine mapping of DNA from this family revealed homozygous alleles in the affected individuals, and a multipoint logarithm of the odds (lod) score of 3.3, within 2.3 mb of chromosome 12q24.33. Previously, Inbal et al. [1995: Am J Med Genet 55:372–378] described siblings with a similar clinical phenotype who lived in Israel but originated from the same Iranian town as the US family. Focused analysis of DNA from the Israeli family confirmed the presence of identical, homozygous alleles in the affected of the US and Israeli families within 1.2 mb of chromosome 12q24.33. Combined multipoint linkage analysis revealed a maximum lod score of 5.41 at the 132 cM position of chromosome 12. Therefore, in these two families of Jewish‐Iranian descent, a disease gene for MSA maps to a 1.2 mb region of chromosome 12q24.33. This region contains 6 well described genes (SFRS8, MMP17, ULK1, PUS1, EP400, and GALNT9) and at least 15 additional putative transcripts. The known genes are expressed in multiple tissues and lack a function specific to mitochondria, making none an obvious candidate. The eventual identification of the disease gene in MSA is expected to provide insight into the tissue specificity and phenotypic variability of mitochondrial disease.