Defects in the IFT-B component IFT172 cause Jeune and Mainzer-Saldino syndromes in humans

Intraflagellar transport (IFT) depends on two evolutionarily conserved modules, subcomplexes A (IFT-A) and B (IFT-B), to drive ciliary assembly and maintenance. All six IFT-A components and their motor protein, DYNC2H1, have been linked to human skeletal ciliopathies, including asphyxiating thoracic dystrophy (ATD; also known as Jeune syndrome), Sensenbrenner syndrome, and Mainzer-Saldino syndrome (MZSDS). Conversely, the 14 subunits in the IFT-B module, with the exception of IFT80, have unknown roles in human disease. To identify additional IFT-B components defective in ciliopathies, we independently performed different mutation analyses: candidate-based sequencing of all IFT-B-encoding genes in 1,467 individuals with a nephronophthisis-related ciliopathy or whole-exome resequencing in 63 individuals with ATD. We thereby detected biallelic mutations in the IFT-B-encoding gene IFT172 in 12 families. All affected individuals displayed abnormalities of the thorax and/or long bones, as well as renal, hepatic, or retinal involvement, consistent with the diagnosis of ATD or MZSDS. Additionally, cerebellar aplasia or hypoplasia characteristic of Joubert syndrome was present in 2 out of 12 families. Fibroblasts from affected individuals showed disturbed ciliary composition, suggesting alteration of ciliary transport and signaling. Knockdown of ift172 in zebrafish recapitulated the human phenotype and demonstrated a genetic interaction between ift172 and ift80. In summary, we have identified defects in IFT172 as a cause of complex ATD and MZSDS. Our findings link the group of skeletal ciliopathies to an additional IFT-B component, IFT172, similar to what has been shown for IFT-A.

Genetic Mechanisms Underlying Autism Spectrum Disorders

Autism is a childhood-onset developmental disorder characterized by deficits in reciprocal social interaction, verbal and non-verbal communication, and dependence on routines and rituals. It belongs to a spectrum of disorders (autism spectrum disorders, ASDs) which share core symptoms but show considerable variation in severity. The whole spectrum affects 0.6-0.7% of children worldwide, inducing a substantial public health burden and causing suffering to the affected families. Despite having a very high heritability, ASDs have shown exceptional genetic heterogeneity, which has complicated the identification of risk variants and left the etiology largely unknown. However, recent studies suggest that rare, family-specific factors contribute significantly to the genetic basis of ASDs. In this study, we investigated the role of DISC1 (Disrupted-in-schizophrenia-1) in ASDs, and identified association with markers and haplotypes previously associated with psychiatric phenotypes. We identified four polymorphic micro-RNA target sites in the 3 UTR of DISC1, and showed that hsa-miR-559 regulates DISC1 expression in vitro in an allele-specific manner. We also analyzed an extended autism pedigree with genealogical roots in Central Finland reaching back to the 17th century. To take advantage of the beneficial characteristics of population isolates to gene mapping and reduced genetic heterogeneity observed in distantly related individuals, we performed a microsatellite-based genome-wide screen for linkage and linkage disequilibrium in this pedigree. We identified a putative autism susceptibility locus on chromosome 19p13.3 and obtained further support for previously reported loci at 1q23 and 15q11-q13. To follow-up these findings, we extended our study sample from the same sub-isolate and initiated a genome-wide analysis of homozygosity and allelic sharing using high-density SNP markers. We identified a small number of haplotypes shared by different subsets of the genealogically connected cases, along with convergent biological pathways from SNP and gene expression data, which highlighted axon guidance molecules in the pathogenesis of ASDs. In conclusion, the results obtained in this thesis show that multiple distinct genetic variants are responsible for the ASD phenotype even within single pedigrees from an isolated population. We suggest that targeted resequencing of the shared haplotypes, linkage regions, and other susceptibility loci is essential to identify the causal variants. We also report a possible micro-RNA mediated regulatory mechanism, which might partially explain the wide-range neurobiological effects of the DISC1 gene.

Associations of interleukin-1 (IL-1) gene variation and IL-1 receptor antagonist phenotypes with immunological responses, metabolic dysregulation and type 2 diabetes

The upstream proinflammatory interleukin-1 (IL-1) cytokines, together with a naturally occurring IL-1 receptor antagonist (IL-1Ra), play a significant role in several diseases and physiologic conditions. The IL-1 proteins affect glucose homeostasis at multiple levels contributing to vascular injuries and metabolic dysregulations that precede diabetes. An association between IL-1 gene variations and IL-1Ra levels has been suggested, and genetic studies have reported associations with metabolic dysregulation and altered inflammatory responses. The principal aims of this study were to: 1) examine the associations of IL-1 gene variation and IL-1Ra expression in the development and persistence of thyroid antibodies in subacute thyroiditis; 2) investigate the associations of common variants in the IL-1 gene family with plasma glucose and insulin concentrations, glucose homeostasis measures and prevalent diabetes in a representative population sample; 3) investigate genetic and non-genetic determinants of IL-1Ra phenotypes in a cross-sectional setting in three independent study populations; 4) investigate in a prospective setting (a) whether variants of the IL-1 gene family are predictors for clinically incident diabetes in two population-based observational cohort studies; and (b) whether the IL-1Ra levels predict the progression of metabolic syndrome to overt diabetes during the median follow-up of 10.8 and 7.1 years. Results from on patients with subacte thyroiditis showed that the systemic IL-1Ra levels are elevated during a specific proinflammatory response and they correlated with C-reactive protein (CRP) levels. Genetic variation in the IL-1 family seemed to have an association with the appearance of thyroid peroxidase antibodies and persisting local autoimmune responses during the follow-up. Analysis of patients suffering from diabetes and metabolic traits suggested that genetic IL-1 variation and IL-1Ra play a role in glucose homeostasis and in the development of type 2 diabetes. The coding IL-1 beta SNP rs1143634 was associated with traits related to insulin resistance in cross-sectional analyses. Two haplotype variants of the IL-1 beta gene were associated with prevalent diabetes or incident diabetes in a prospective setting and both of these haplotypes were tagged by rs1143634. Three variants of the IL-1Ra gene and one of the IL-1 beta gene were consistently identified as significant, independent determinants of the IL-1Ra phenotype in two or three populations. The proportion of the phenotypic variation explained by the genetic factors was modest however, while obesity and other metabolic traits explained a larger part. Body mass index was the strongest predictor of systemic IL-1Ra concentration overall. Furthermore, the age-adjusted IL-1Ra concentrations were elevated in individuals with metabolic syndrome or diabetes when compared to those free of metabolic dysregulation. In prospective analyses the systemic IL-1Ra levels were found as independent predictors for the development of diabetes in people with metabolic syndrome even after adjustment for multiple other factors, including plasma glucose and CRP levels. The predictive power of IL-1Ra was better than that of CRP. The prospective results also provided some evidence for a role of common IL-1 alpha promoter SNP rs1800587 in the development of type 2 diabetes among men and suggested that the role may be gender specific. Likewise, common variations in the IL-1 beta coding region may have a gender specific association with diabetes development. Further research on the potential benefits of IL-1Ra measurements in identifying individuals at high risk for diabetes, who then could be targeted for specific treatment interventions, is warranted. It has been reported in the recent literature that IL-1Ra secreted from adipose tissue has beneficial effects on glucose homeostasis. Furthermore, treatment with recombinant human IL-1Ra has been shown to have a substantial therapeutic potential. The genetic results from the prospective analyses performed in this study remain inconclusive, but together with the cross-sectional analyses they suggest gender-specific effects of the IL-1 variants on the risk of diabetes. Larger studies with more extensive genotyping and resequencing may help to pinpoint the exact variants responsible and to further elucidate the biological mechanisms for the observed associations. This would improve our understanding of the pathways linking inflammation and obesity with glucose and insulin metabolism.

RECQL4 and p53 potentiate the activity of polymerase and maintain the integrity of the human mitochondrial genome

Germline mutations in RECQL4 and p53 lead to cancer predisposition syndromes, Rothmund-Thomson syndrome (RTS) and Li-Fraumeni syndrome (LFS), respectively. RECQL4 is essential for the transport of p53 to the mitochondria under unstressed conditions. Here, we show that both RECQL4 and p53 interact with mitochondrial polymerase (Pol gamma A/B2) and regulate its binding to the mitochondrial DNA (mtDNA) control region (D-loop). Both RECQL4 and p53 bind to the exonuclease and polymerase domains of Pol gamma A. Kinetic constants for interactions between Pol gamma A-RECQL4, Pol gamma A-p53 and Pol gamma B-p53 indicate that RECQL4 and p53 are accessory factors for Pol gamma A-Pol gamma B and Pol gamma A-DNA interactions. RECQL4 enhances the binding of Pol gamma A to DNA, thereby potentiating the exonuclease and polymerization activities of Pol gamma A/B2. To investigate whether lack of RECQL4 and p53 results in increased mitochondrial genome instability, resequencing of the entire mitochondrial genome was undertaken from multiple RTS and LFS patient fibroblasts. We found multiple somatic mutations and polymorphisms in both RTS and LFS patient cells. A significant number of mutations and polymorphisms were common between RTS and LFS patients. These changes are associated with either aging and/or cancer, thereby indicating that the phenotypes associated with these syndromes may be due to deregulation of mitochondrial genome stability caused by the lack of RECQL4 and p53. Summary: The biochemical mechanisms by which RECQL4 and p53 affect mtDNA replication have been elucidated. Resequencing of RTS and LFS patients' mitochondrial genome reveals common mutations indicating similar mechanisms of regulation by RECQL4 and p53.

Whole exome sequencing in an Indian family links Coats plus syndrome and dextrocardia with a homozygous novel CTC1 and a rare HES7 variation

Background: Coats plus syndrome is an autosomal recessive, pleiotropic, multisystem disorder characterized by retinal telangiectasia and exudates, intracranial calcification with leukoencephalopathy and brain cysts, osteopenia with predisposition to fractures, bone marrow suppression, gastrointestinal bleeding and portal hypertension. It is caused by compound heterozygous mutations in the CTC1 gene. Case presentation: We encountered a case of an eight-year old boy from an Indian family with manifestations of Coats plus syndrome along with an unusual occurrence of dextrocardia and situs inversus. Targeted resequencing of the CTC1 gene as well as whole exome sequencing (WES) were conducted in this family to identify the causal variations. The identified candidate variations were screened in ethnicity matched healthy controls. The effect of CTC1 variation on telomere length was assessed using Southern blot. A novel homozygous missense mutation c.1451A > C (p.H484P) in exon 9 of the CTC1 gene and a rare 3'UTR known dbSNP variation (c.*556 T > C) in HES7 were identified as the plausible candidates associated with this complex phenotype of Coats plus and dextrocardia. This CTC1 variation was absent in the controls and we also observed a reduced telomere length in the affected individual's DNA, suggesting its likely pathogenic nature. The reported p.H484P mutation is located in the N-terminal 700 amino acid regionthat is important for the binding of CTC1 to ssDNA through its two OB domains. WES data also showed a rare homozygous missense variation in the TEK gene in the affected individual. Both HES7 and TEK are targets of the Notch signaling pathway. Conclusions: This is the first report of a genetically confirmed case of Coats plus syndrome from India. By means of WES, the genetic variations in this family with unique and rare complex phenotype could be traced effectively. We speculate the important role of Notch signaling in this complex phenotypic presentation of Coats plus syndrome and dextrocardia. The present finding will be useful for genetic diagnosis and carrier detection in the family and for other patients with similar disease manifestations.

The Interplay between Natural Selection and Susceptibility to Melanoma on Allele 374F of SLC45A2 Gene in a South European Population

We aimed to study the selective pressures interacting on SLC45A2 to investigate the interplay between selection and susceptibility to disease. Thus, we enrolled 500 volunteers from a geographically limited population (Basques from the North of Spain) and by resequencing the whole coding region and intron 5 of the 34 most and the 34 least pigmented individuals according to the reflectance distribution, we observed that the polymorphism Leu374Phe (L374F, rs16891982) was statistically associated with skin color variability within this sample. In particular, allele 374F was significantly more frequent among the individuals with lighter skin. Further genotyping an independent set of 558 individuals of a geographically wider population with known ancestry in the Spanish population also revealed that the frequency of L374F was significantly correlated with the incident UV radiation intensity. Selection tests suggest that allele 374F is being positively selected in South Europeans, thus indicating that depigmentation is an adaptive process. Interestingly, by genotyping 119 melanoma samples, we show that this variant is also associated with an increased susceptibility to melanoma in our populations. The ultimate driving force for this adaptation is unknown, but it is compatible with the vitamin D hypothesis. This shows that molecular evolution analysis can be used as a useful technology to predict phenotypic and biomedical consequences in humans.

Phantom mutation hotspots in human mitochondrial DNA

Phantom mutations are systematic artifacts generated in the course of the sequencing process. Contra common belief these artificial mutations are nearly ubiquitous in sequencing results, albeit at frequencies that may vary dramatically. The amount of artifacts depends not only on the sort of automated sequencer and sequencing chemistry employed, but also on other lab-specific factors. An experimental study executed on four samples under various combinations of sequencing conditions revealed a number of phantom mutations occurring at the same sites of mitochondrial DNA (mtDNA) repeatedly. To confirm these and identify further hotspots for artifacts, > 5000 mtDNA electropherograms were screened for artificial patterns. Further, > 30000 published hypervariable segment 1 sequences were compared at potential hotspots for phantom mutations, especially for variation at positions 16085 and 16197. Resequencing of several samples confirmed the artificial nature of these and other polymorphisms in the original publications. Single-strand sequencing, as typically executed in medical and anthropological studies, is thus highly vulnerable to this kind of artifacts. In particular, phantom mutation hotspots could easily lead to misidentification of somatic mutations and to misinterpretations in all kinds of clinical mtDNA studies.

Rare variants create synthetic genome-wide associations.

Genome-wide association studies (GWAS) have now identified at least 2,000 common variants that appear associated with common diseases or related traits (http://www.genome.gov/gwastudies), hundreds of which have been convincingly replicated. It is generally thought that the associated markers reflect the effect of a nearby common (minor allele frequency >0.05) causal site, which is associated with the marker, leading to extensive resequencing efforts to find causal sites. We propose as an alternative explanation that variants much less common than the associated one may create "synthetic associations" by occurring, stochastically, more often in association with one of the alleles at the common site versus the other allele. Although synthetic associations are an obvious theoretical possibility, they have never been systematically explored as a possible explanation for GWAS findings. Here, we use simple computer simulations to show the conditions under which such synthetic associations will arise and how they may be recognized. We show that they are not only possible, but inevitable, and that under simple but reasonable genetic models, they are likely to account for or contribute to many of the recently identified signals reported in genome-wide association studies. We also illustrate the behavior of synthetic associations in real datasets by showing that rare causal mutations responsible for both hearing loss and sickle cell anemia create genome-wide significant synthetic associations, in the latter case extending over a 2.5-Mb interval encompassing scores of "blocks" of associated variants. In conclusion, uncommon or rare genetic variants can easily create synthetic associations that are credited to common variants, and this possibility requires careful consideration in the interpretation and follow up of GWAS signals.

Host genetics and HIV-1: the final phase?

This is a crucial transition time for human genetics in general, and for HIV host genetics in particular. After years of equivocal results from candidate gene analyses, several genome-wide association studies have been published that looked at plasma viral load or disease progression. Results from other studies that used various large-scale approaches (siRNA screens, transcriptome or proteome analysis, comparative genomics) have also shed new light on retroviral pathogenesis. However, most of the inter-individual variability in response to HIV-1 infection remains to be explained: genome resequencing and systems biology approaches are now required to progress toward a better understanding of the complex interactions between HIV-1 and its human host.

Hybrid error correction and de novo assembly of single-molecule sequencing reads.

Single-molecule sequencing instruments can generate multikilobase sequences with the potential to greatly improve genome and transcriptome assembly. However, the error rates of single-molecule reads are high, which has limited their use thus far to resequencing bacteria. To address this limitation, we introduce a correction algorithm and assembly strategy that uses short, high-fidelity sequences to correct the error in single-molecule sequences. We demonstrate the utility of this approach on reads generated by a PacBio RS instrument from phage, prokaryotic and eukaryotic whole genomes, including the previously unsequenced genome of the parrot Melopsittacus undulatus, as well as for RNA-Seq reads of the corn (Zea mays) transcriptome. Our long-read correction achieves >99.9% base-call accuracy, leading to substantially better assemblies than current sequencing strategies: in the best example, the median contig size was quintupled relative to high-coverage, second-generation assemblies. Greater gains are predicted if read lengths continue to increase, including the prospect of single-contig bacterial chromosome assembly.

Mitochondrial mutations in adenoid cystic carcinoma of the salivary glands.

BACKGROUND: The MitoChip v2.0 resequencing array is an array-based technique allowing for accurate and complete sequencing of the mitochondrial genome. No studies have investigated mitochondrial mutation in salivary gland adenoid cystic carcinomas. METHODOLOGY: The entire mitochondrial genome of 22 salivary gland adenoid cystic carcinomas (ACC) of salivary glands and matched leukocyte DNA was sequenced to determine the frequency and distribution of mitochondrial mutations in ACC tumors. PRINCIPAL FINDINGS: Seventeen of 22 ACCs (77%) carried mitochondrial mutations, ranging in number from 1 to 37 mutations. A disproportionate number of mutations occurred in the D-loop. Twelve of 17 tumors (70.6%) carried mutations resulting in amino acid changes of translated proteins. Nine of 17 tumors (52.9%) with a mutation carried an amino acid changing mutation in the nicotinamide adenine dinucleotide dehydrogenase (NADH) complex. CONCLUSIONS/SIGNIFICANCE: Mitochondrial mutation is frequent in salivary ACCs. The high incidence of amino acid changing mutations implicates alterations in aerobic respiration in ACC carcinogenesis. D-loop mutations are of unclear significance, but may be associated with alterations in transcription or replication.

Investigation of DNA polymorphisms in SMAD genes for genetic predisposition to diabetic nephropathy in patients with type 1 diabetes mellitus

Aims/hypothesis: SMAD proteins are involved in multiple signalling pathways and are key modulators of gene expression. We hypothesised that genetic variation in selected SMAD genes contributes to susceptibility to diabetic nephropathy. Methods: We selected 13 haplotype tag (ht) single nucleotide polymorphisms (SNPs) from 67 variants identified by resequencing the SMAD2 and SMAD3 genes. For SMAD1, SMAD4 and SMAD5 genes, genotype data were downloaded for 217 SNPs from Phase II of the International HapMap project. Of these, 85 SNPs met our inclusion criteria, resulting in the selection of 13 tag SNPs for further investigation. A case-control approach was employed, using 267 nephropathic patients and 442 controls with type 1 diabetes from Ireland. Two further populations (totalling 1,407 patients, 2,238 controls) were genotyped to validate initial findings. Genotyping was conducted using iPLEX, TaqMan and gel electrophoresis.
Results: The distribution of genotypes was in Hardy-Weinberg equilibrium. Analysis by the ? 2 test of genotype and allele frequencies in patients versus controls in the Irish population (n?=?709) revealed evidence for the association of one allele at 5% level of significance (rs10515478, p uncorrected?=?0.006; p corrected?=?0.04). This finding represents a relatively small difference in allele frequency of 6.4% in the patient group compared with 10.7% in the control group; this difference was not supported in subsequent investigations using DNA from European individuals with similar phenotypic characteristics.
Conclusions/interpretation: We selected an appropriate subset of variants for the investigation of common genetic risk factors and assessed SMAD1 to SMAD5 genes for association with diabetic nephropathy. We conclude that common polymorphisms in these genes do not strongly influence genetic susceptibility to diabetic nephropathy in white individuals with type 1 diabetes mellitus.

Development of a Diagnostic Genetic Test for Simplex and Autosomal Recessive Retinitis Pigmentosa

PURPOSE: Retinitis pigmentosa (RP) causes hereditary blindness in adults (prevalence, approximately 1 in 4000). Each of the more than 30 causative genes identified to date are responsible for only a small percentage of cases. Genetic diagnosis via traditional methods is problematic, and a single test with a higher probability of detecting the causative mutation would be very beneficial for the clinician. The goal of this study therefore was to develop a high-throughput screen capable of detecting both known mutations and novel mutations within all genes implicated in autosomal recessive or simplex RP. DESIGN: Evaluation of diagnostic technology. PARTICIPANTS AND CONTROLS: Participants were 56 simplex and autosomal recessive RP patients, with 360 population controls unscreened for ophthalmic disease. METHODS: A custom genechip capable of resequencing all exons containing known mutations in 19 disease-associated genes was developed (RP genechip). A second, commercially available arrayed primer extension (APEX) system was used to screen 501 individual previously reported variants. The ability of these high-throughput approaches to identify pathogenic variants was assessed in a cohort of simplex and autosomal recessive RP patients. MAIN OUTCOME MEASURES: Number of mutations and potentially pathogenic variants identified. RESULTS: The RP genechip identified 44 sequence variants: 5 previously reported mutations; 22 known single nucleotide polymorphisms (SNPs); 11 novel, potentially pathogenic variants; and 6 novel SNPs. There was strong concordance with the APEX array, but only the RP genechip detected novel variants. For example, identification of a novel mutation in CRB1 revealed a patient, who also had a single previously known CRB1 mutation, to be a compound heterozygote. In some individuals, potentially pathogenic variants were discovered in more than one gene, consistent with the existence of disease modifier effects resulting from mutations at a second locus. CONCLUSIONS: The RP genechip provides the significant advantage of detecting novel variants and could be expected to detect at least one pathogenic variant in more than 50% of patients. The APEX array provides a reliable method to detect known pathogenic variants in autosomal recessive RP and simplex RP patients and is commercially available. High-throughput genotyping for RP is evolving into a clinically useful genetic diagnostic tool.

Genomewide linkage scan of schizophrenia in a large multicenter pedigree sample using single nucleotide polymorphisms

A genomewide linkage scan was carried out in eight clinical samples of informative schizophrenia families. After all quality control checks, the analysis of 707 European-ancestry families included 1615 affected and 1602 unaffected genotyped individuals, and the analysis of all 807 families included 1900 affected and 1839 unaffected individuals. Multipoint linkage analysis with correction for marker-marker linkage disequilibrium was carried out with 5861 single nucleotide polymorphisms (SNPs; Illumina version 4.0 linkage map). Suggestive evidence for linkage ( European families) was observed on chromosomes 8p21, 8q24.1, 9q34 and 12q24.1 in nonparametric and/or parametric analyses. In a logistic regression allele-sharing analysis of linkage allowing for intersite heterogeneity, genomewide significant evidence for linkage was observed on chromosome 10p12. Significant heterogeneity was also observed on chromosome 22q11.1. Evidence for linkage across family sets and analyses was most consistent on chromosome 8p21, with a one-LOD support interval that does not include the candidate gene NRG1, suggesting that one or more other susceptibility loci might exist in the region. In this era of genomewide association and deep resequencing studies, consensus linkage regions deserve continued attention, given that linkage signals can be produced by many types of genomic variation, including any combination of multiple common or rare SNPs or copy number variants in a region. Molecular Psychiatry (2009) 14, 786-795; doi:10.1038/mp.2009.11; published online 17 February 2009

A Candidate Gene Association Study Identifies DAPL1 as a Female-Specific Susceptibility Locus for Age-Related Macular Degeneration (AMD)

Age-related macular degeneration (AMD) is the leading cause of blindness among white caucasians over the age of 50 years with a prevalence rate expected to increase markedly with an anticipated increase in the life span of the world population. To further expand our knowledge of the genetic architecture of the disease, we pursued a candidate gene approach assessing 25 genes and a total of 109 variants. Of these, synonymous single nucleotide polymorphism (SNP) rs17810398 located in death-associated protein-like 1 (DAPL1) was found to be associated with AMD in a joint analysis of 3,229 cases and 2,835 controls from five studies [combined P ADJ = 1.15 × 10(-6), OR 1.332 (1.187-1.496)]. This association was characterized by a highly significant sex difference (P diff = 0.0032) in that it was clearly confined to females with genome-wide significance [P ADJ = 2.62 × 10(-8), OR 1.541 (1.324-1.796); males: P ADJ = 0.382, OR 1.084 (0.905-1.298)]. By targeted resequencing of risk and non-risk associated haplotypes in the DAPL1 locus, we identified additional potentially functional risk variants, namely a common 897-bp deletion and a SNP predicted to affect a putative binding site of an exonic splicing enhancer. We show that the risk haplotype correlates with a reduced retinal transcript level of two, less frequent, non-canonical DAPL1 isoforms. DAPL1 plays a role in epithelial differentiation and may be involved in apoptotic processes thereby suggesting a possible novel pathway in AMSaveD pathogenesis.