Fedik Rahimov

Disruption of an AP-2α binding site in an IRF6 enhancer is associated with cleft lip

Previously we have shown that nonsyndromic cleft lip with or without cleft palate (NSCL/P) is strongly associated with SNPs in IRF6 (interferon regulatory factor 6). Here, we use multispecies sequence comparisons to identify a common SNP (rs642961, G>A) in a newly identified IRF6 enhancer. The A allele is significantly overtransmitted (P = 1 × 10−11) in families with NSCL/P, in particular those with cleft lip but not cleft palate. Further, there is a dosage effect of the A allele, with a relative risk for cleft lip of 1.68 for the AG genotype and 2.40 for the AA genotype. EMSA and ChIP assays demonstrate that the risk allele disrupts the binding site of transcription factor AP-2α and expression analysis in the mouse localizes the enhancer activity to craniofacial and limb structures. Our findings place IRF6 and AP-2α in the same developmental pathway and identify a high-frequency variant in a regulatory element contributing substantially to a common, complex disorder.

Genetics of nonsyndromic orofacial clefts.

With an average worldwide prevalence of approximately 1.2/1000 live births, orofacial clefts are the most common craniofacial birth defects in humans. Like other complex disorders, these birth defects are thought to result from the complex interplay of multiple genes and environmental factors. Significant progress in the identification of underlying genes and pathways has benefited from large populations available for study, increased international collaboration, rapid advances in genotyping technology, and major improvements in analytic approaches. Here we review recent advances in genetic epidemiological approaches to complex traits and their applications to studies of nonsyndromic orofacial clefts. Our main aim is to bring together a discussion of new and previously identified candidate genes to create a more cohesive picture of interacting pathways that shape the human craniofacial region. In future directions, we highlight the need to search for copy number variants that affect gene dosage and rare variants that are possibly associated with a higher disease penetrance. In addition, sequencing of protein-coding regions in candidate genes and screening for genetic variation in noncoding regulatory elements will help advance this important area of research.

Cellular and molecular mechanisms underlying muscular dystrophy

The muscular dystrophies are a group of heterogeneous genetic diseases characterized by progressive degeneration and weakness of skeletal muscle. Since the discovery of the first muscular dystrophy gene encoding dystrophin, a large number of genes have been identified that are involved in various muscle-wasting and neuromuscular disorders. Human genetic studies complemented by animal model systems have substantially contributed to our understanding of the molecular pathomechanisms underlying muscle degeneration. Moreover, these studies have revealed distinct molecular and cellular mechanisms that link genetic mutations to diverse muscle wasting phenotypes.

Genetic variants in IRF6 and the risk of facial clefts: single-marker and haplotype-based analyses in a population-based case-control study of facial clefts in Norway.

Mutations in the gene encoding interferon regulatory factor 6 (IRF6) underlie a common form of syndromic clefting known as Van der Woude syndrome. Lip pits and missing teeth are the only additional features distinguishing the syndrome from isolated clefts. Van der Woude syndrome, therefore, provides an excellent model for studying the isolated forms of clefting. From a population‐based case‐control study of facial clefts in Norway (1996–2001), we selected 377 cleft lip with or without cleft palate (CL/P), 196 cleft palate only (CPO), and 763 control infant‐parent triads for analysis. We genotyped six single nucleotide polymorphisms within the IRF6 locus and estimated the relative risks (RR) conferred on the child by alleles and haplotypes of the child and of the mother. On the whole, there were strong statistical associations with CL/P but not CPO in our data. In single‐marker analyses, mothers with a double‐dose of the ‘a’‐allele at rs4844880 had an increased risk of having a child with CL/P (RR=1.85, 95% confidence interval: 1.04–3.25; P=0.036). An RR of 0.38 (95% confidence interval: 0.16–0.92; P=0.031) was obtained when the child carried a single‐dose of the ‘a’‐allele at rs2235371 (the p.V274I polymorphism). The P‐value for the overall test was <0.001. In haplotype analyses, several of the fetal and maternal haplotype relative risks were statistically significant individually but were not strong enough to show up on the overall test (P=0.113). Taken together, these findings further support a role for IRF6 variants in clefting of the lip and provide specific risk estimates in a Norwegian population. Genet. Epidemiol. 2008.

Telomere position effect regulates DUX4 in human facioscapulohumeral muscular dystrophy

Telomeres may regulate human disease by at least two independent mechanisms. First, replicative senescence occurs once short telomeres generate DNA-damage signals that produce a barrier to tumor progression. Second, telomere position effects (TPE) could change gene expression at intermediate telomere lengths in cultured human cells. Here we report that telomere length may contribute to the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a late-onset disease genetically residing only 25–60 kilobases from the end of chromosome 4q. We used a floxable telomerase to generate isogenic clones with different telomere lengths from affected patients and their unaffected siblings. DUX4, the primary candidate for FSHD pathogenesis, is upregulated over ten-fold in FSHD myoblasts and myotubes with short telomeres, and its expression is inversely proportional to telomere length. FSHD may be the first known human disease in which TPE contributes to age-related phenotype.

Transcriptional profiling in facioscapulohumeral muscular dystrophy to identify candidate biomarkers.

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive neuromuscular disorder caused by contractions of repetitive elements within the macrosatellite D4Z4 on chromosome 4q35. The pathophysiology of FSHD is unknown and, as a result, there is currently no effective treatment available for this disease. To better understand the pathophysiology of FSHD and develop mRNA-based biomarkers of affected muscles, we compared global analysis of gene expression in two distinct muscles obtained from a large number of FSHD subjects and their unaffected first-degree relatives. Gene expression in two muscle types was analyzed using GeneChip Gene 1.0 ST arrays: biceps, which typically shows an early and severe disease involvement; and deltoid, which is relatively uninvolved. For both muscle types, the expression differences were mild: using relaxed cutoffs for differential expression (fold change ≥1.2; nominal P value <0.01), we identified 191 and 110 genes differentially expressed between affected and control samples of biceps and deltoid muscle tissues, respectively, with 29 genes in common. Controlling for a false-discovery rate of <0.25 reduced the number of differentially expressed genes in biceps to 188 and in deltoid to 7. Expression levels of 15 genes altered in this study were used as a “molecular signature” in a validation study of an additional 26 subjects and predicted them as FSHD or control with 90% accuracy based on biceps and 80% accuracy based on deltoids.

MicroRNA-486–dependent modulation of DOCK3/PTEN/AKT signaling pathways improves muscular dystrophy–associated symptoms

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, which results in dysfunctional signaling pathways within muscle. Previously, we identified microRNA-486 (miR-486) as a muscle-enriched microRNA that is markedly reduced in the muscles of dystrophin-deficient mice (Dmdmdx-5Cv mice) and in DMD patient muscles. Here, we determined that muscle-specific transgenic overexpression of miR-486 in muscle of Dmdmdx-5Cv mice results in reduced serum creatine kinase levels, improved sarcolemmal integrity, fewer centralized myonuclei, increased myofiber size, and improved muscle physiology and performance. Additionally, we identified dedicator of cytokinesis 3 (DOCK3) as a miR-486 target in skeletal muscle and determined that DOCK3 expression is induced in dystrophic muscles. DOCK3 overexpression in human myotubes modulated PTEN/AKT signaling, which regulates muscle hypertrophy and growth, and induced apoptosis. Furthermore, several components of the PTEN/AKT pathway were markedly modulated by miR-486 in dystrophin-deficient muscle. Skeletal muscle-specific miR-486 overexpression in Dmdmdx-5Cv animals decreased levels of DOCK3, reduced PTEN expression, and subsequently increased levels of phosphorylated AKT, which resulted in an overall beneficial effect. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates the disease progression of dystrophin-deficient skeletal muscle.

GLI2 mutations in four Brazilian patients: How wide is the phenotypic spectrum?†

We report four patients with GLI2 mutations together with their associated phenotypes: (1) holoprosencephaly‐like phenotype, (2) anophthalmia, branchial arch anomalies, and CNS abnormalities, (3) heminasal aplasia and orbital anomalies, and (4) lobar holoprosencephaly. This diversity of phenotypes expands our understanding. Findings include not only (1) holoprosencephaly or a holoprosencephaly‐like phenotype, but also (2) heminasal aplasia with orbital anomalies, and (3) branchial arch anomalies of the type seen in hemifacial microsomia with anophthalmia and in oculoauriculofrontonasal syndrome. Finally, this is the first report of a double mutation involving GLI2 and PTCH in the same patient.

Gene expression profiling of skeletal muscles treated with a soluble activin type IIB receptor

Inhibition of the myostatin signaling pathway is emerging as a promising therapeutic means to treat muscle wasting and degenerative disorders. Activin type IIB receptor (ActRIIB) is the putative myostatin receptor, and a soluble activin receptor (ActRIIB-Fc) has been demonstrated to potently inhibit a subset of transforming growth factor (TGF)-β family members including myostatin. To determine reliable and valid biomarkers for ActRIIB-Fc treatment, we assessed gene expression profiles for quadriceps muscles from mice treated with ActRIIB-Fc compared with mice genetically lacking myostatin and control mice. Expression of 134 genes was significantly altered in mice treated with ActRIIB-Fc over a 2-wk period relative to control mice (fold change > 1.5, P < 0.001), whereas the number of significantly altered genes in mice treated for 2 days was 38, demonstrating a time-dependent response to ActRIIB-Fc in overall muscle gene expression. The number of significantly altered genes in Mstn(-/-) mice relative to control mice was substantially higher (360), but for most of these genes the expression levels in the 2-wk treated mice were closer to the levels in the Mstn(-/-) mice than in control mice (P < 10⁻³⁰). Expression levels of 30 selected genes were further validated with quantitative real-time polymerase chain reaction (qPCR), and a correlation of ≥ 0.89 was observed between the fold changes from the microarray analysis and the qPCR analysis. These data suggest that treatment with ActRIIB-Fc results in overlapping but distinct gene expression signatures compared with myostatin genetic mutation. Differentially expressed genes identified in this study can be used as potential biomarkers for ActRIIB-Fc treatment, which is currently in clinical trials as a therapeutic agent for muscle wasting and degenerative disorders.

Comparative RNA editing in autistic and neurotypical cerebella.

Adenosine-to-inosine (A-to-I) RNA editing is a neurodevelopmentally regulated epigenetic modification shown to modulate complex behavior in animals. Little is known about human A-to-I editing, but it is thought to constitute one of many molecular mechanisms connecting environmental stimuli and behavioral outputs. Thus, comprehensive exploration of A-to-I RNA editing in human brains may shed light on gene–environment interactions underlying complex behavior in health and disease. Synaptic function is a main target of A-to-I editing, which can selectively recode key amino acids in synaptic genes, directly altering synaptic strength and duration in response to environmental signals. Here, we performed a high-resolution survey of synaptic A-to-I RNA editing in a human population, and examined how it varies in autism, a neurodevelopmental disorder in which synaptic abnormalities are a common finding. Using ultra-deep (>1000 × ) sequencing, we quantified the levels of A-to-I editing of 10 synaptic genes in postmortem cerebella from 14 neurotypical and 11 autistic individuals. A high dynamic range of editing levels was detected across individuals and editing sites, from 99.6% to below detection limits. In most sites, the extreme ends of the population editing distributions were individuals with autism. Editing was correlated with isoform usage, clusters of correlated sites were identified, and differential editing patterns examined. Finally, a dysfunctional form of the editing enzyme adenosine deaminase acting on RNA B1 was found more commonly in postmortem cerebella from individuals with autism. These results provide a population-level, high-resolution view of A-to-I RNA editing in human cerebella and suggest that A-to-I editing of synaptic genes may be informative for assessing the epigenetic risk for autism.