Severe phenotype with cis-acting heterozygous PMP22 mutations

We report on a 20-year-old male with severe Charcot-Marie-Tooth (CMT) disease and a de novo deletion (c.281delG, p.G94AfsX17) on the paternal PMP22 allele harboring c.353C>T (p.T118M). RNA-based sequence analysis confirmed the absence of nonsense-mediated decay and the presence of the mutant transcripts in Epstein-Barr virus-transformed lymphoblastoid cells of our patient. His clinical findings included early onset of polyneuropathy, loss of muscle mass with distal pareses, hammer toes, and progressive scoliosis. There was no neuropsychological alteration. Our results suggest that the deletion c.281delG alone is responsible for the severe CMT phenotype. To the best of our knowledge, this is the second report on a proven paternal origin of a de novo single-base mutation in the PMP22 gene.

A MIF haplotype is associated with the outcome of patients with severe sepsis: a case control study

BACKGROUND: Macrophage migration inhibitory factor (MIF) plays an important regulatory role in sepsis. In the promoter region a C/G single nucleotide polymorphism (SNP) at position -173 (rs755622) and a CATT5-8 microsatellite at position -794 are related to modified promoter activity. The purpose of the study was to analyze their association with the incidence and outcome of severe sepsis. METHODS: Genotype distributions and allele frequencies in 169 patients with severe sepsis, 94 healthy blood donors and 183 postoperative patients without signs of infection or inflammation were analyzed by real time PCR and Sequence analysis. All included individuals were Caucasians. RESULTS: Genotype distribution and allele frequencies of severe sepsis patients were comparable to both control groups. However, the genotype and allele frequencies of both polymorphisms were associated significantly with the outcome of severe sepsis. The highest risk of dying from severe sepsis was detectable in patients carrying a haplotype with the alleles -173 C and CATT7 (p = 0.0005, fisher exact test, RR = 1,806, CI: 1.337 to 2.439). CONCLUSION: The haplotype with the combination of the -173 C allele and the -794 CATT7 allele may not serve as a marker for susceptibility to sepsis, but may help identify septic patients at risk of dying.

Quantitative sequence analysis of FBN1 premature termination codons provides evidence for incomplete NMD in leukocytes

We improved, evaluated, and used Sanger sequencing for quantification of single nucleotide polymorphism (SNP) variants in transcripts and gDNA samples. This improved assay resulted in highly reproducible relative allele frequencies (e.g., for a heterozygous gDNA 50.0+/-1.4%, and for a missense mutation-bearing transcript 46.9+/-3.7%) with a lower detection limit of 3-9%. It provided excellent accuracy and linear correlation between expected and observed relative allele frequencies. This sequencing assay, which can also be used for the quantification of copy number variations (CNVs), methylations, mosaicisms, and DNA pools, enabled us to analyze transcripts of the FBN1 gene in fibroblasts and blood samples of patients with suspected Marfan syndrome not only qualitatively but also quantitatively. We report a total of 18 novel and 19 known FBN1 sequence variants leading to a premature termination codon (PTC), 26 of which we analyzed by quantitative sequencing both at gDNA and cDNA levels. The relative amounts of PTC-containing FBN1 transcripts in fresh and PAXgene-stabilized blood samples were significantly higher (33.0+/-3.9% to 80.0+/-7.2%) than those detected in affected fibroblasts with inhibition of nonsense-mediated mRNA decay (NMD) (11.0+/-2.1% to 25.0+/-1.8%), whereas in fibroblasts without NMD inhibition no mutant alleles could be detected. These results provide evidence for incomplete NMD in leukocytes and have particular importance for RNA-based analyses not only in FBN1 but also in other genes.

A frameshift mutation in the cubilin gene (CUBN) in Border Collies with Imerslund-Gräsbeck syndrome (selective cobalamin malabsorption).

Imerslund-Gräsbeck syndrome (IGS) or selective cobalamin malabsorption has been described in humans and dogs. IGS occurs in Border Collies and is inherited as a monogenic autosomal recessive trait in this breed. Using 7 IGS cases and 7 non-affected controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 3.53 Mb interval on chromosome 2. We re-sequenced the genome of one affected dog at ∼10× coverage and detected 17 non-synonymous variants in the critical interval. Two of these non-synonymous variants were in the cubilin gene (CUBN), which is known to play an essential role in cobalamin uptake from the ileum. We tested these two CUBN variants for association with IGS in larger cohorts of dogs and found that only one of them was perfectly associated with the phenotype. This variant, a single base pair deletion (c.8392delC), is predicted to cause a frameshift and premature stop codon in the CUBN gene. The resulting mutant open reading frame is 821 codons shorter than the wildtype open reading frame (p.Q2798Rfs*3). Interestingly, we observed an additional nonsense mutation in the MRC1 gene encoding the mannose receptor, C type 1, which was in perfect linkage disequilibrium with the CUBN frameshift mutation. Based on our genetic data and the known role of CUBN for cobalamin uptake we conclude that the identified CUBN frameshift mutation is most likely causative for IGS in Border Collies.

Comparative genetic analyses point to HCP5 as susceptibility locus for HCV-associated hepatocellular carcinoma

BACKGROUND & AIMS: Recently, genetic variations in MICA (lead single nucleotide polymorphism [SNP] rs2596542) were identified by a genome-wide association study (GWAS) to be associated with hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC) in Japanese patients. In the present study, we sought to determine whether this SNP is predictive of HCC development in the Caucasian population as well. METHODS: An extended region around rs2596542 was genotyped in 1924 HCV-infected patients from the Swiss Hepatitis C Cohort Study (SCCS). Pair-wise correlation between key SNPs was calculated both in the Japanese and European populations (HapMap3: CEU and JPT). RESULTS: To our surprise, the minor allele A of rs2596542 in proximity of MICA appeared to have a protective impact on HCC development in Caucasians, which represents an inverse association as compared to the one observed in the Japanese population. Detailed fine-mapping analyses revealed a new SNP in HCP5 (rs2244546) upstream of MICA as strong predictor of HCV-related HCC in the SCCS (univariable p=0.027; multivariable p=0.0002, odds ratio=3.96, 95% confidence interval=1.90-8.27). This newly identified SNP had a similarly directed effect on HCC in both Caucasian and Japanese populations, suggesting that rs2244546 may better tag a putative true variant than the originally identified SNPs. CONCLUSIONS: Our data confirms the MICA/HCP5 region as susceptibility locus for HCV-related HCC and identifies rs2244546 in HCP5 as a novel tagging SNP. In addition, our data exemplify the need for conducting meta-analyses of cohorts of different ethnicities in order to fine map GWAS signals.

Multiple Hypersensitivities Including Recurrent Airway Obstruction, Insect Bite Hypersensitivity, and Urticaria in 2 Warmblood Horse Populations.

BACKGROUND Multiple hypersensitivities (MHS) have been described in humans, cats, and dogs, but not horses. HYPOTHESES Horses suffering from recurrent airway obstruction (RAO), insect bite hypersensitivity (IBH), or urticaria (URT) will have an increased risk of also being affected by another one of these hypersensitivities. This predisposition for MHS also will be associated with decreased shedding of strongylid eggs in feces and with a single nucleotide polymorphism (SNP BIEC2-224511), previously shown to be associated with RAO. ANIMALS The first population (P1) included 119 randomly sampled horses representative of the Swiss sporthorse population; the replication population (P2) included 210 RAO-affected Warmblood horses and 264 RAO-unaffected controls. All horses were Warmbloods, 14 years or older. METHODS Associations between disease phenotypes (RAO, IBH, URT, MHS) fecal egg counts, the SNP BIEC2-224511 as well as management and environmental factors were investigated. RESULTS In P1, RAO-affected horses had a 13.1 times higher odds ratio (OR) of also suffering from IBH (P = .004). In P2, the respective OR was 7.4 (P = .002) and IBH-affected horses also showed a 7.1 times increased OR of concomitantly suffering from URT (P < .001). IBH, URT, and MHS phenotypes were significantly associated with the absence of nematode eggs in the feces. CONCLUSIONS AND CLINICAL IMPORTANCE This is the first report of MHS in horses. Specifically, an increased risk for IBH should be expected in RAO-affected horses.

SNPs for parentage testing and traceability in globally diverse breeds of sheep

DNA-based parentage determination accelerates genetic improvement in sheep by increasing pedigree accuracy. Single nucleotide polymorphism (SNP) markers can be used for determining parentage and to provide unique molecular identifiers for tracing sheep products to their source. However, the utility of a particular "parentage SNP" varies by breed depending on its minor allele frequency (MAF) and its sequence context. Our aims were to identify parentage SNPs with exceptional qualities for use in globally diverse breeds and to develop a subset for use in North American sheep. Starting with genotypes from 2,915 sheep and 74 breed groups provided by the International Sheep Genomics Consortium (ISGC), we analyzed 47,693 autosomal SNPs by multiple criteria and selected 163 with desirable properties for parentage testing. On average, each of the 163 SNPs was highly informative (MAF≥0.3) in 48±5 breed groups. Nearby polymorphisms that could otherwise confound genetic testing were identified by whole genome and Sanger sequencing of 166 sheep from 54 breed groups. A genetic test with 109 of the 163 parentage SNPs was developed for matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The scoring rates and accuracies for these 109 SNPs were greater than 99% in a panel of North American sheep. In a blinded set of 96 families (sire, dam, and non-identical twin lambs), each parent of every lamb was identified without using the other parent's genotype. In 74 ISGC breed groups, the median estimates for probability of a coincidental match between two animals (PI), and the fraction of potential adults excluded from parentage (PE) were 1.1×10(-39) and 0.999987, respectively, for the 109 SNPs combined. The availability of a well-characterized set of 163 parentage SNPs facilitates the development of high-throughput genetic technologies for implementing accurate and economical parentage testing and traceability in many of the world's sheep breeds.

A Deletion in the VLDLR Gene in Eurasier Dogs with Cerebellar Hypoplasia Resembling a Dandy-Walker-Like Malformation (DWLM).

Dandy-Walker-like malformation (DWLM) is the result of aberrant brain development and mainly characterized by cerebellar hypoplasia. DWLM affected dogs display a non-progressive cerebellar ataxia. Several DWLM cases were recently observed in the Eurasier dog breed, which strongly suggested a monogenic autosomal recessive inheritance in this breed. We performed a genome-wide association study (GWAS) with 9 cases and 11 controls and found the best association of DWLM with markers on chromosome 1. Subsequent homozygosity mapping confirmed that all 9 cases were homozygous for a shared haplotype in this region, which delineated a critical interval of 3.35 Mb. We sequenced the genome of an affected Eurasier and compared it with the Boxer reference genome and 47 control genomes of dogs from other breeds. This analysis revealed 4 private non-synonymous variants in the critical interval of the affected Eurasier. We genotyped these variants in additional dogs and found perfect association for only one of these variants, a single base deletion in the VLDLR gene encoding the very low density lipoprotein receptor. This variant, VLDLR:c.1713delC is predicted to cause a frameshift and premature stop codon (p.W572Gfs*10). Variants in the VLDLR gene have been shown to cause congenital cerebellar ataxia and mental retardation in human patients and Vldlr knockout mice also display an ataxia phenotype. Our combined genetic data together with the functional knowledge on the VLDLR gene from other species thus strongly suggest that VLDLR:c.1713delC is indeed causing DWLM in Eurasier dogs.

Association of a common vitamin D-binding protein polymorphism with inflammatory bowel disease

Inflammatory bowel diseases (IBDs), Crohn's disease, and ulcerative colitis (UC), are multifactorial disorders, characterized by chronic inflammation of the intestine. A number of genetic components have been proposed to contribute to IBD pathogenesis. In this case-control study, we investigated the association between two common vitamin D-binding protein (DBP) genetic variants and IBD susceptibility. These two single nucleotide polymorphisms (SNPs) in exon 11 of the DBP gene, at codons 416 (GAT>GAG; Asp>Glu) and 420 (ACG>AAG; Thr>Lys), have been previously suggested to play roles in the etiology of other autoimmune diseases.

Association of kynurenine aminotransferase II gene C401T polymorphism with immune response in patients with meningitis

The kynurenine (KYN) pathway has been shown to be altered in several diseases which compromise the central nervous system (CNS) including infectious diseases such as bacterial meningitis (BM). The aim of this study was to assess single nucleotide polymorphisms (SNPs) in four genes of KYN pathway in patients with meningitis and their correlation with markers of immune response in BM.

Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine

The synthesis of a caged RNA phosphoramidite building block containing the oxidatively damaged base 5-hydroxycytidine (5-HOrC) has been accomplished. To determine the effect of this highly mutagenic lesion on complementary base recognition and coding properties, this building block was incorporated into a 12-mer oligoribonucleotide for Tm and CD measurements and a 31-mer template strand for primer extension experiments with HIV-, AMV- and MMLV-reverse transcriptase (RT). In UV-melting experiments, we find an unusual biphasic transition with two distinct Tm's when 5-HOrC is paired against a DNA or RNA complement with the base guanine in opposing position. The higher Tm closely matches that of a C-G base pair while the lower is close to that of a C-A mismatch. In single nucleotide extension reactions, we find substantial misincorporation of dAMP and to a lesser extent dTMP, with dAMP almost equaling that of the parent dGMP in the case of HIV-RT. A working hypothesis for the biphasic melting transition does not invoke tautomeric variability of 5-HOrC but rather local structural perturbations of the base pair at low temperature induced by interactions of the 5-HO group with the phosphate backbone. The properties of this RNA damage is discussed in the context of its putative biological function.

A polymorphism within the equine CRISP3 gene is associated with stallion fertility in Hanoverian warmblood horses

Fertility of stallions is of high economic importance, especially for large breeding organisations and studs. Breeding schemes with respect to fertility traits and selection of stallions at an early stage may be improved by including molecular genetic markers associated with traits. The genes coding for equine cysteine-rich secretory proteins (CRISPs) are promising candidate genes because previous studies have shown that CRISPs play a role in the fertilising ability of male animals. We have previously characterised the three equine CRISP genes and identified a non-synonymous polymorphism in the CRISP1 gene. In this study, we report one non-synonymous polymorphism in the CRISP2 gene and four non-synonymous polymorphisms in the CRISP3 gene. All six CRISP polymorphisms were genotyped in 107 Hanoverian breeding stallions. Insemination records of stallions were used to analyse the association between CRISP polymorphisms and fertility traits. Three statistical models were used to evaluate the influence of single mutations, genotypes and haplotypes of the polymorphisms. The CRISP3 AJ459965:c.+622G>A SNP leading to the amino acid substitution E208K was significantly associated with the fertility of stallions. Stallions heterozygous for the CRISP3 c.+622G>A SNP had lower fertility than homozygous stallions (P = 0.0234). The pregnancy rate per cycle in these stallions was estimated to be approximately 7% lower than in stallions homozygous at this position.

Polymorphism and chromosomal location of the MC4R (melanocortin-4 receptor) gene in the dog and red fox

The melanocortin-4 receptor (MC4R) is expressed in the hypothalamus and regulates energy intake and body weight. In silico screening of the canine chromosome 1 sequence and a comparison with the porcine MC4R sequence by BLAST were performed. The nucleotide sequence of the whole coding region and 3'- and 5'-flanking regions of the dog (1214 bp) and red fox (1177 bp) MC4R gene was established and high conservation of the nucleotide sequences was revealed (99%). Five sets of PCR primers were designed and a search for polymorphism was performed by the SSCP technique in a group of 31 dogs representing nineteen breeds and 35 farm red foxes. Sequencing of DNA fragments, representing the identified SSCP patterns, revealed three single nucleotide polymorphisms (including a missense one) in dogs and four silent SNPs in red foxes. An average SNP frequency was approx. 1/400 bp in the dog and 1/300 bp in the red fox. We mapped the MC4R gene by FISH to the canine chromosome 1 (CFA1q1.1) and to the red fox chromosome 5 (VVU5p1.2).

No role of matrixmetalloproteinase-3 genetic promoter polymorphism 1171 as a risk factor for cirrhosis in alcoholic liver disease

BACKGROUND: As only a minority of alcoholics develop cirrhosis, polymorphic genes, whose products are involved in fibrosis development were suggested to confer individual susceptibility. We tested whether a functional promoter polymorphism in the gene encoding matrix metalloproteinase-3 (MMP-3; 1171 5A/6A) was associated liver cirrhosis in alcoholics. METHODS: Independent cohorts from the UK and Germany were studied. (i) UK cohort: 320 alcoholic cirrhotics and 183 heavy drinkers without liver damage and (ii) German cohort: 149 alcoholic cirrhotics, 220 alcoholic cirrhotics who underwent liver transplantation and 151 alcoholics without liver disease. Patients were genotyped for MMP-3 variants by restriction fragment length polymorphism, single strand confirmation polymorphism, and direct sequencing. In addition, MMP-3 transcript levels were correlated with MMP-3 genotype in normal liver tissues. RESULTS: Matrix metalloproteinase-3 genotype and allele distribution in all 1023 alcoholic patients were in Hardy-Weinberg equilibrium. No significant differences in MMP-3 genotype and allele frequencies were observed either between alcoholics with or without cirrhosis. There were no differences in hepatic mRNA transcription levels according to MMP-3 genotype. CONCLUSIONS: Matrix metalloproteinase-3 1171 promoter polymorphism plays no role in the genetic predisposition for liver cirrhosis in alcoholics. Stringently designed candidate gene association studies are required to exclude chance observations.

Citrate- vs. acetate-based dialysate in bicarbonate haemodialysis: consequences on haemodynamics, coagulation, acid-base status, and electrolytes

BACKGROUND: A concentrate for bicarbonate haemodialysis acidified with citrate instead of acetate has been marketed in recent years. The small amount of citrate used (one-fifth of the concentration adopted in regional anticoagulation) protects against intradialyser clotting while minimally affecting the calcium concentration. The aim of this study was to compare the impact of citrate- and acetate-based dialysates on systemic haemodynamics, coagulation, acid-base status, calcium balance and dialysis efficiency. METHODS: In 25 patients who underwent a total of 375 dialysis sessions, an acetate dialysate (A) was compared with a citrate dialysate with (C+) or without (C) calcium supplementation (0.25 mmol/L) in a randomised single-blind cross-over study. Systemic haemodynamics were evaluated using pulse-wave analysis. Coagulation, acid-base status, calcium balance and dialysis efficiency were assessed using standard biochemical markers. RESULTS: Patients receiving the citrate dialysate had significantly lower systolic blood pressure (BP) (-4.3 mmHg, p < 0.01) and peripheral resistances (PR) (-51 dyne.sec.cm-5, p < 0.001) while stroke volume was not increased. In hypertensive patients there was a substantial reduction in BP (-7.8 mmHg, p < 0.01). With the C+ dialysate the BP gap was less pronounced but the reduction in PR was even greater (-226 dyne.sec.cm-5, p < 0.001). Analyses of the fluctuations in PR and of subjective tolerance suggested improved haemodynamic stability with the citrate dialysate. Furthermore, an increase in pre-dialysis bicarbonate and a decrease in pre-dialysis BUN, post-dialysis phosphate and ionised calcium were noted. Systemic coagulation activation was not influenced by citrate. CONCLUSION: The positive impact on dialysis efficiency, acid-base status and haemodynamics, as well as the subjective tolerance, together indicate that citrate dialysate can significantly contribute to improving haemodialysis in selected patients.