Verification of CRISPR editing and finding transgenic inserts by Xdrop indirect sequence capture followed by short- and long-read sequencing.

Validation of CRISPR-Cas9 editing typically explores the immediate vicinity of the gene editing site and distal off-target sequences, which has led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kilobases-long and only affect one allele. The gold standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach allows the detection of small editing events but fails in detecting large rearrangements, in particular when only one allele is affected. Detection of large rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we implemented Xdrop™, a new microfluidic technology that allows targeted enrichment of long regions (~100 kb) using just a single standard PCR primer set. Sequencing of the enriched CRISPR-Cas9 gene-edited region in four cell lines on long- and short-read sequencing platforms unravelled unknown and unintended genome editing events. The analysis revealed accidental kilobases-large insertions in three of the cell lines, which remained undetected using standard procedures. We also applied the targeted enrichment approach to identify the integration site of a transgene in a mouse line. The results demonstrate the potential of this technology in gene editing validation as well as in more classic transgenics.

Novel insights into the disease transcriptome of human diabetic glomeruli and tubulointerstitium.

BACKGROUND: Diabetic nephropathy (DN) is the most common cause of end-stage renal disease, affecting ∼30% of the rapidly growing diabetic population, and strongly associated with cardiovascular risk. Despite this, the molecular mechanisms of disease remain unknown. METHODS: RNA sequencing (RNAseq) was performed on paired, micro-dissected glomerular and tubulointerstitial tissue from patients diagnosed with DN [n = 19, 15 males, median (range) age: 61 (30-85) years, chronic kidney disease stages 1-4] and living kidney donors [n = 20, 12 males, median (range) age: 56 (30-70) years]. RESULTS: Principal component analysis showed a clear separation between glomeruli and tubulointerstitium transcriptomes. Differential expression analysis identified 1550 and 4530 differentially expressed genes, respectively (adjusted P < 0.01). Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses highlighted activation of inflammation and extracellular matrix (ECM) organization pathways in glomeruli, and immune and apoptosis pathways in tubulointerstitium of DN patients. Specific gene modules were associated with renal function in weighted gene co-expression network analysis. Increased messengerRNA (mRNA) expression of renal damage markers lipocalin 2 (LCN) and hepatitis A virus cellular receptor1 (HAVCR1) in the tubulointerstitial fraction was observed alongside higher urinary concentrations of the corresponding proteins neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in DN patients. CONCLUSIONS: Here we present the first RNAseq experiment performed on paired glomerular and tubulointerstitial samples from DN patients. We show that prominent disease-specific changes occur in both compartments, including relevant cellular processes such as reorganization of ECM and inflammation (glomeruli) as well as apoptosis (tubulointerstitium). The results emphasize the potential of utilizing high-throughput transcriptomics to decipher disease pathways and treatment targets in this high-risk patient population.

Two variants on chromosome 17 confer prostate cancer risk, and the one in TCF2 protects against type 2 diabetes.

We performed a genome-wide association scan to search for sequence variants conferring risk of prostate cancer using 1,501 Icelandic men with prostate cancer and 11,290 controls. Follow-up studies involving three additional case-control groups replicated an association of two variants on chromosome 17 with the disease. These two variants, 33 Mb apart, fall within a region previously implicated by family-based linkage studies on prostate cancer. The risks conferred by these variants are moderate individually (allele odds ratio of about 1.20), but because they are common, their joint population attributable risk is substantial. One of the variants is in TCF2 (HNF1beta), a gene known to be mutated in individuals with maturity-onset diabetes of the young type 5. Results from eight case-control groups, including one West African and one Chinese, demonstrate that this variant confers protection against type 2 diabetes.

Genome-wide association study identifies a second prostate cancer susceptibility variant at 8q24.

Prostate cancer is the most prevalent noncutaneous cancer in males in developed regions, with African American men having among the highest worldwide incidence and mortality rates. Here we report a second genetic variant in the 8q24 region that, in conjunction with another variant we recently discovered, accounts for about 11%-13% of prostate cancer cases in individuals of European descent and 31% of cases in African Americans. We made the current discovery through a genome-wide association scan of 1,453 affected Icelandic individuals and 3,064 controls using the Illumina HumanHap300 BeadChip followed by four replication studies. A key step in the discovery was the construction of a 14-SNP haplotype that efficiently tags a relatively uncommon (2%-4%) susceptibility variant in individuals of European descent that happens to be very common (approximately 42%) in African Americans. The newly identified variant shows a stronger association with affected individuals who have an earlier age at diagnosis.

A common variant associated with prostate cancer in European and African populations.

With the increasing incidence of prostate cancer, identifying common genetic variants that confer risk of the disease is important. Here we report such a variant on chromosome 8q24, a region initially identified through a study of Icelandic families. Allele -8 of the microsatellite DG8S737 was associated with prostate cancer in three case-control series of European ancestry from Iceland, Sweden and the US. The estimated odds ratio (OR) of the allele is 1.62 (P = 2.7 x 10(-11)). About 19% of affected men and 13% of the general population carry at least one copy, yielding a population attributable risk (PAR) of approximately 8%. The association was also replicated in an African American case-control group with a similar OR, in which 41% of affected individuals and 30% of the population are carriers. This leads to a greater estimated PAR (16%) that may contribute to higher incidence of prostate cancer in African American men than in men of European ancestry.

The prognostic value of microRNA-126 and microvessel density in patients with stage II colon cancer: results from a population cohort.

BACKGROUND: Angiogenesis plays a pivotal role in malignant tumour growth and the metastatic process. We analysed the prognostic value of two angiogenesis parameters, microRNA-126 (miRNA-126) and microvessel density (MVD), in a population based cohort of patients operated for stage II colon cancer. METHODS: A total of 560 patients were included. Analyses were performed on formalin fixed paraffin embedded tissue from the primary tumours. The analysis of miRNA-126 expression was performed by qPCR. Microvessels were visualised by CD105 and quantified in hot spots using a light microscope. The analyses were correlated with recurrence-free cancer specific survival (RF-CSS) and overall survival (OS). RESULTS: Low miRNA-126 expression was significantly correlated to T4, high malignancy grade, tumour perforation, fixation, and the presence of microsatellite instability. A prognostic impact on OS was detected in the simple analysis favouring patients with high miRNA-126 expression p = 0.03, and borderline significance as to RF-CSS, p = 0.08. The impact on OS demonstrated borderline significance in a following multiple Cox regression analysis, hazard ratio 0.76 (95% confidence interval, 0.58-1.00), p = 0.051. The MVD estimate was not associated with either RF-CSS, p = 0.49, or OS, p = 0.94. CONCLUSION: The current population based study of patients operated for stage II colon cancer demonstrated correlations between several prognostic unfavourable characteristics and miRNA-126 and argues for a possible prognostic impact on overall survival. An influence on survival by the MVD estimate was not detected.

Assessing sample and miRNA profile quality in serum and plasma or other biofluids.

MicroRNAs (miRNAs) constitute a class of small cellular RNAs (typically 21-23nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, although they are relatively few in number (less than 2000 human miRNAs). The high relative stability of miRNA in common clinical tissues and biofluids (e.g. plasma, serum, urine, saliva, etc.) and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNA quantification as a promising new tool for a wide range of diagnostic applications. Furthermore miRNAs have been shown to be rapidly released from tissues into the circulation with the development of pathology. To facilitate discovery and clinical development of miRNA-based biomarkers, we developed a genome-wide Locked Nucleic Acid (LNA™)-based miRNA qPCR platform with unparalleled sensitivity and robustness. The platform allows high-throughput profiling of miRNAs from important clinical sources without the need for pre-amplification. Using this system, we have profiled thousands of biofluid samples including blood derived plasma and serum. An extensive quality control (QC) system has been implemented in order to secure technical excellence and reveal any unwanted bias coming from pre-analytical or analytical variables. We present our approaches to sample and RNA QC as well as data QC and normalization. Specifically we have developed normal reference ranges for circulating miRNAs in serum and plasma as well as a hemolysis indicator based on microRNA expression.

Large recurrent microdeletions associated with schizophrenia.

Reduced fecundity, associated with severe mental disorders, places negative selection pressure on risk alleles and may explain, in part, why common variants have not been found that confer risk of disorders such as autism, schizophrenia and mental retardation. Thus, rare variants may account for a larger fraction of the overall genetic risk than previously assumed. In contrast to rare single nucleotide mutations, rare copy number variations (CNVs) can be detected using genome-wide single nucleotide polymorphism arrays. This has led to the identification of CNVs associated with mental retardation and autism. In a genome-wide search for CNVs associating with schizophrenia, we used a population-based sample to identify de novo CNVs by analysing 9,878 transmissions from parents to offspring. The 66 de novo CNVs identified were tested for association in a sample of 1,433 schizophrenia cases and 33,250 controls. Three deletions at 1q21.1, 15q11.2 and 15q13.3 showing nominal association with schizophrenia in the first sample (phase I) were followed up in a second sample of 3,285 cases and 7,951 controls (phase II). All three deletions significantly associate with schizophrenia and related psychoses in the combined sample. The identification of these rare, recurrent risk variants, having occurred independently in multiple founders and being subject to negative selection, is important in itself. CNV analysis may also point the way to the identification of additional and more prevalent risk variants in genes and pathways involved in schizophrenia.

Variants conferring risk of atrial fibrillation on chromosome 4q25.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in humans and is characterized by chaotic electrical activity of the atria. It affects one in ten individuals over the age of 80 years, causes significant morbidity and is an independent predictor of mortality. Recent studies have provided evidence of a genetic contribution to AF. Mutations in potassium-channel genes have been associated with familial AF but account for only a small fraction of all cases of AF. We have performed a genome-wide association scan, followed by replication studies in three populations of European descent and a Chinese population from Hong Kong and find a strong association between two sequence variants on chromosome 4q25 and AF. Here we show that about 35% of individuals of European descent have at least one of the variants and that the risk of AF increases by 1.72 and 1.39 per copy. The association with the stronger variant is replicated in the Chinese population, where it is carried by 75% of individuals and the risk of AF is increased by 1.42 per copy. A stronger association was observed in individuals with typical atrial flutter. Both variants are adjacent to PITX2, which is known to have a critical function in left-right asymmetry of the heart.

Analysis of a gene panel for targeted sequencing of colorectal cancer samples.

Colorectal cancer (CRC) is a leading cause of death worldwide. Surgical intervention is a successful treatment for stage I patients, whereas other more advanced cases may require adjuvant chemotherapy. The selection of effective adjuvant treatments remains, however, challenging. Accurate patient stratification is necessary for the identification of the subset of patients likely responding to treatment, while sparing others from pernicious treatment. Targeted sequencing approaches may help in this regard, enabling rapid genetic investigation, and at the same time easily applicable in routine diagnosis. We propose a set of guidelines for the identification, including variant calling and filtering, of somatic mutations driving tumorigenesis in the absence of matched healthy tissue. We also discuss the inclusion criteria for the generation of our gene panel. Furthermore, we evaluate the prognostic impact of individual genes, using Cox regression models in the context of overall survival and disease-free survival. These analyses confirmed the role of commonly used biomarkers, and shed light on controversial genes such as CYP2C8. Applying those guidelines, we created a novel gene panel to investigate the onset and progression of CRC in 273 patients. Our comprehensive biomarker set includes 266 genes that may play a role in the progression through the different stages of the disease. Tracing the developmental state of the tumour, and its resistances, is instrumental in patient stratification and reliable decision making in precision clinical practice.

Isolation and characterization of a thermostable RNA ligase 1 from a Thermus scotoductus bacteriophage TS2126 with good single-stranded DNA ligation properties.

We have recently sequenced the genome of a novel thermophilic bacteriophage designated as TS2126 that infects the thermophilic eubacterium Thermus scotoductus. One of the annotated open reading frames (ORFs) shows homology to T4 RNA ligase 1, an enzyme of great importance in molecular biology, owing to its ability to ligate single-stranded nucleic acids. The ORF was cloned, and recombinant protein was expressed, purified and characterized. The recombinant enzyme ligates single-stranded nucleic acids in an ATP-dependent manner and is moderately thermostable. The recombinant enzyme exhibits extremely high activity and high ligation efficiency. It can be used for various molecular biology applications including RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The TS2126 RNA ligase catalyzed both inter- and intra-molecular single-stranded DNA ligation to >50% completion in a matter of hours at an elevated temperature, although favoring intra-molecular ligation on RNA and single-stranded DNA substrates. The properties of TS2126 RNA ligase 1 makes it very attractive for processes like adaptor ligation, and single-stranded solid phase gene synthesis.

Quantitative RT-PCR for MicroRNAs in Biofluids.

MicroRNAs in biofluids hold great promise as minimally invasive diagnostic biomarkers for a wide range of diseases and biological processes. One of the most sensitive technologies for detection and measuring expression levels of microRNA is quantitative RT-PCR. However, quantification of microRNA in biofluid samples is challenging in many ways. Biofluids contain low levels of RNA and high levels of inhibitors of enzymatic processes like reverse transcription and PCR. Furthermore, biofluids are susceptible to many preanalytical variables. Here we describe procedures developed to address these challenges, which include highly sensitive and accurate microRNA detection methods, combined with optimized protocols for sample handling and preparation, and extensive quality control (QC) procedures.

Genome-Wide Comparison of Next-Generation Sequencing and qPCR Platforms for microRNA Profiling in Serum.

This study compares next-generation sequencing (NGS) technologies that have been optimized specifically for biofluid samples, with more established qPCR-based methods for profiling microRNAs in biofluids. The same patient serum samples were analyzed by NGS and qPCR, and differences in the serum microRNA profile between HBV and HCV infected patients were investigated. While there was overall good agreement between NGS and qPCR, there were some differences between the platforms, highlighting the importance of validation.

Discovery and characterization of a thermostable bacteriophage RNA ligase homologous to T4 RNA ligase 1.

Thermophilic viruses represent a novel source of genetic material and enzymes with great potential for use in biotechnology. We have isolated a number of thermophilic viruses from geothermal areas in Iceland, and by combining high throughput genome sequencing and state of the art bioinformatics we have identified a number of genes with potential use in biotechnology. We have also demonstrated the existence of thermostable counterparts of previously known bacteriophage enzymes. Here we describe a thermostable RNA ligase 1 from the thermophilic bacteriophage RM378 that infects the thermophilic eubacterium Rhodothermus marinus. The RM378 RNA ligase 1 has a temperature optimum of 60-64 degrees C and it ligates both RNA and single-stranded DNA. Its thermostability and ability to work under conditions of high temperature where nucleic acid secondary structures are removed makes it an ideal enzyme for RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), and other RNA and DNA ligation applications.

A novel MALDI-TOF based methodology for genotyping single nucleotide polymorphisms.

A new MALDI-TOF based detection assay was developed for analysis of single nucleotide polymorphisms (SNPs). It is a significant modification on the classic three-step minisequencing method, which includes a polymerase chain reaction (PCR), removal of excess nucleotides and primers, followed by primer extension in the presence of dideoxynucleotides using modified thermostable DNA polymerase. The key feature of this novel assay is reliance upon deoxynucleotide mixes, lacking one of the nucleotides at the polymorphic position. During primer extension in the presence of depleted nucleotide mixes, standard thermostable DNA polymerases dissociate from the template at positions requiring a depleted nucleotide; this principal was harnessed to create a genotyping assay. The assay design requires a primer- extension primer having its 3'-end one nucleotide upstream from the interrogated site. The assay further utilizes the same DNA polymerase in both PCR and the primer extension step. This not only simplifies the assay but also greatly reduces the cost per genotype compared to minisequencing methodology. We demonstrate accurate genotyping using this methodology for two SNPs run in both singleplex and duplex reactions. We term this assay nucleotide depletion genotyping (NUDGE). Nucleotide depletion genotyping could be extended to other genotyping assays based on primer extension such as detection by gel or capillary electrophoresis.

Differential expression of plasma miRNAs in patients with unprovoked venous thromboembolism and healthy control individuals.

BACKGROUND: Venous thromboembolism (VTE) remains the third most common cardiovascular disease with a vague pathogenesis. Circulating miRNAs are small regulatory RNAs found in plasma, serum and other body fluids in an apparently stable form. Although circulating miRNAs, a novel family of regulatory molecules, emerge as a promising class of biomarkers in many cardiovascular diseases and malignancies, knowledge on plasma miRNA levels in VTE remains sparse. AIMS: The present work was conducted as a pilot study in order to estimate the plasma levels of miRNAs in patients with unprovoked VTE and to assess miRNAs as potential novel biomarkers of VTE. METHODS: Twenty patients with a history of unprovoked VTE 1-5 years prior to inclusion in the study and twenty age- and sex-matched healthy control participants were enrolled in a case-control study (Tromsø IV). Plasma levels of 742 miRNAs were assessed after RNA extraction and reverse transcription. Profiling of miRNA was conducted on the Universal RT microRNA PCR Human panels I and II (Exiqon, Denmark). For normalization of the data, the average of the assays detected in all samples (n=40 samples) was applied. RESULTS: Ninety-seven miRNAs were detected throughout all samples. Of these, miR-10b-5p, -320a, -320b, -424-5p, and -423-5p were upregulated, whereas miR-103a-3p, -191-5p, -301a-3p, and 199b-3p were downregulated in plasmas of VTE patients versus controls (P≤0.05). These miRNAs were confined to the extracellular vesicles-depleted plasma fraction, and yielded clear clustering distinguishing samples from the VTE and control groups. CONCLUSIONS: The results of this pilot study indicate that plasma miRNAs profiling can provide novel biomarkers of unprovoked VTE.

Efficient identification of miRNAs for classification of tumor origin.

Carcinomas of unknown primary origin constitute 3% to 5% of all newly diagnosed metastatic cancers, with the primary source difficult to classify with current histological methods. Effective cancer treatment depends on early and accurate identification of the tumor; patients with metastases of unknown origin have poor prognosis and short survival. Because miRNA expression is highly tissue specific, the miRNA profile of a metastasis may be used to identify its origin. We therefore evaluated the potential of miRNA profiling to identify the primary tumor of known metastases. Two hundred eight formalin-fixed, paraffin-embedded samples, representing 15 different histologies, were profiled on a locked nucleic acid-enhanced microarray platform, which allows for highly sensitive and specific detection of miRNA. On the basis of these data, we developed and cross-validated a novel classification algorithm, least absolute shrinkage and selection operator, which had an overall accuracy of 85% (CI, 79%-89%). When the classifier was applied on an independent test set of 48 metastases, the primary site was correctly identified in 42 cases (88% accuracy; CI, 75%-94%). Our findings suggest that miRNA expression profiling on paraffin tissue can efficiently predict the primary origin of a tumor and may provide pathologists with a molecular diagnostic tool that can improve their capability to correctly identify the origin of hitherto unidentifiable metastatic tumors and, eventually, enable tailored therapy.

A serum microRNA signature is associated with the immune control of chronic hepatitis B virus infection.

BACKGROUND AND AIMS: The virus/host interplay mediates liver pathology in chronic HBV infection. MiRNAs play a pivotal role in virus/host interactions and are detected in both serum and HBsAg-particles, but studies of their dynamics during chronic infection and antiviral therapy are missing. We studied serum miRNAs during different phases of chronic HBV infection and antiviral treatment. METHODS: MiRNAs were profiled by miRCURY-LNA-Universal-RT-miRNA-PCR (Exiqon-A/S) and qPCR-panels-I/II-739-miRNA-assays and single-RT-q-PCRs. Two cohorts of well-characterized HBsAg-carriers were studied (median follow-up 34-52 months): a) training-panel (141 sera) and HBsAg-particles (32 samples) from 61 HBsAg-carriers and b) validation-panel (136 sera) from 84 carriers. RESULTS: Thirty-one miRNAs were differentially expressed in inactive-carriers (IC) and chronic-hepatitis-B (CHB) with the largest difference for miR-122-5p, miR-99a-5p and miR-192-5p (liver-specific-miRNAs), over-expressed in both sera and HBsAg-particles of CHB (ANOVA/U-test p-values: <0.000001/0.000001; <0.000001/0.000003; <0.000001/0.000005, respectively) and significantly down-regulated during- and after-treatment in sustained-virological-responders (SVR). MiRNA-profiles of IC and SVR clustered in the heatmap. Liver-miRNAs were combined with miR-335, miR-126 and miR-320a (internal controls) to build a MiR-B-Index with 100% sensitivity, 83.3% and 92.5% specificity (-1.7 cut-off) in both training and validation cohorts to identify IC. MiR-B-Index (-5.72, -20.43/14.38) correlated with ALT (49, 10/2056 U/l, ρ = -0.497, p<0.001), HBV-DNA (4.58, undetectable/>8.3 Log10 IU/mL, ρ = -0.732, p<0.001) and HBsAg (3.40, 0.11/5.49 Log10 IU/mL, ρ = -0.883, p<0.001). At multivariate analysis HBV-DNA (p = 0.002), HBsAg (p<0.001) and infection-phase (p<0.001), but not ALT (p = 0.360) correlated with MiR-B-Index. In SVR to Peg-IFN/NUCs MiR-B-Index improved during-therapy and post-treatment reaching IC-like values (5.32, -1.65/10.91 vs 6.68, 0.54/9.53, p = 0.324) beckoning sustained HBV-immune-control earlier than HBsAg-decline. CONCLUSIONS: Serum miRNA profile change dynamically during the different phases of chronic HBV infection. We identified a miRNA signature associated with both natural-occurring and therapy-induced immune control of HBV infection. The MiR-B-Index might be a useful biomarker for the early identification of the sustained switch from CHB to inactive HBV-infection in patients treated with antivirals.

A sequence variant on 17q21 is associated with age at onset and severity of asthma.

A sequence variant (rs7216389-T) near the ORMDL3 gene on chromosome 17q21 was recently found to be associated with childhood asthma. We sought to evaluate the effect of rs7216389-T on asthma subphenotypes and its correlation with expression levels of neighboring genes. The association of rs7216389-T with asthma was replicated in six European and one Asian study cohort (N=4917 cases N=34 589 controls). In addition, we found that the association of rs7216389-T was confined to cases with early onset of asthma, particularly in early childhood (age: 0-5 years OR=1.51, P=6.89.10(-9)) and adolescence (age: 14-17 years OR=1.71, P=5.47.10(-9)). A weaker association was observed for onset between 6 and 13 years of age (OR=1.17, P=0.035), but none for adult-onset asthma (OR=1.07, P=0.12). Cases were further stratified by sex, asthma severity and atopy status. An association with greater asthma severity was observed among early-onset asthma cases (P=0.0012), but no association with sex or atopy status was observed among the asthma cases. An association between sequence variants and the expression of genes in the 17q21 region was assessed in white blood cell RNA samples collected from Icelandic individuals (n=743). rs7216389 associated with the expression of GSDMB and ORMDL3 genes. However, other sequence variants showing a weaker association with asthma compared with that of rs7216389 were more strongly associated with the expression of both genes. Thus, the contribution of rs7216389-T to the development of asthma is unlikely to operate only through an impact on the expression of ORMDL3 or GSDMB genes.

Common variants on 9q22.33 and 14q13.3 predispose to thyroid cancer in European populations.

In order to search for sequence variants conferring risk of thyroid cancer we conducted a genome-wide association study in 192 and 37,196 Icelandic cases and controls, respectively, followed by a replication study in individuals of European descent. Here we show that two common variants, located on 9q22.33 and 14q13.3, are associated with the disease. Overall, the strongest association signals were observed for rs965513 on 9q22.33 (OR = 1.75; P = 1.7 x 10(-27)) and rs944289 on 14q13.3 (OR = 1.37; P = 2.0 x 10(-9)). The gene nearest to the 9q22.33 locus is FOXE1 (TTF2) and NKX2-1 (TTF1) is among the genes located at the 14q13.3 locus. Both variants contribute to an increased risk of both papillary and follicular thyroid cancer. Approximately 3.7% of individuals are homozygous for both variants, and their estimated risk of thyroid cancer is 5.7-fold greater than that of noncarriers. In a study on a large sample set from the general population, both risk alleles are associated with low concentrations of thyroid stimulating hormone (TSH), and the 9q22.33 allele is associated with low concentration of thyroxin (T(4)) and high concentration of triiodothyronine (T(3)).