8p23 beta-defensin copy number determination by single-locus pseudogene-based paralog ratio tests risk bias due to low-frequency sequence variations.

BACKGROUND The copy number variation (CNV) in beta-defensin genes (DEFB) on human chromosome 8p23 has been proposed to contribute to the phenotypic differences in inflammatory diseases. However, determination of exact DEFB CN is a major challenge in association studies. Quantitative real-time PCR (qPCR), paralog ratio tests (PRT) and multiplex ligation-dependent probe amplification (MLPA) have been extensively used to determine DEFB CN in different laboratories, but inter-method inconsistencies were observed frequently. In this study we asked which one is superior among the three methods for DEFB CN determination. RESULTS We developed a clustering approach for MLPA and PRT to statistically correlate data from a single experiment. Then we compared qPCR, a newly designed PRT and MLPA for DEFB CN determination in 285 DNA samples. We found MLPA had the best convergence and clustering results of the raw data and the highest call rate. In addition, the concordance rates between MLPA or PRT and qPCR (32.12% and 37.99%, respectively) were unacceptably low with underestimated CN by qPCR. Concordance rate between MLPA and PRT (90.52%) was high but PRT systematically underestimated CN by one in a subset of samples. In these samples a sequence variant which caused complete PCR dropout of the respective DEFB cluster copies was found in one primer binding site of one of the targeted paralogous pseudogenes. CONCLUSION MLPA is superior to PRT and even more to qPCR for DEFB CN determination. Although the applied PRT provides in most cases reliable results, such a test is particularly sensitive to low-frequency sequence variations preferably accumulating in loci like pseudogenes which are most likely not under selective pressure. In the light of the superior performance of multiplex assays, the drawbacks of such single PRTs could be overcome by combining more test markers.

Lethal chondrodysplasia in a family of Holstein cattle is associated with a de novo splice site variant of COL2A1

Background Lethal chondrodysplasia (bulldog syndrome) is a well-known congenital syndrome in cattle and occurs sporadically in many breeds. In 2015, it was noticed that about 12 % of the offspring of the phenotypically normal Danish Holstein sire VH Cadiz Captivo showed chondrodysplasia resembling previously reported bulldog calves. Pedigree analysis of affected calves did not display obvious inbreeding to a common ancestor, suggesting the causative allele was not a rare recessive. The normal phenotype of the sire suggested a dominant inheritance with incomplete penetrance or a mosaic mutation. Results Three malformed calves were examined by necropsy, histopathology, radiology, and computed tomography scanning. These calves were morphologically similar and displayed severe disproportionate dwarfism and reduced body weight. The syndrome was characterized by shortening and compression of the body due to reduced length of the spine and the long bones of the limbs. The vicerocranium had severe dysplasia and palatoschisis. The bones had small irregular diaphyses and enlarged epiphyses consisting only of chondroid tissue. The sire and a total of four affected half-sib offspring and their dams were genotyped with the BovineHD SNP array to map the defect in the genome. Significant genetic linkage was obtained for several regions of the bovine genome including chromosome 5 where whole genome sequencing of an affected calf revealed a COL2A1 point mutation (g.32473300 G > A). This private sequence variant was predicted to affect splicing as it altered the conserved splice donor sequence GT at the 5’-end of COL2A1 intron 36, which was changed to AT. All five available cases carried the mutant allele in heterozygous state and all five dams were homozygous wild type. The sire VH Cadiz Captivo was shown to be a gonadal and somatic mosaic as assessed by the presence of the mutant allele at levels of about 5 % in peripheral blood and 15 % in semen. Conclusions The phenotypic and genetic findings are comparable to a previously reported COL2A1 missense mutation underlying lethal chondrodysplasia in the offspring of a mosaic French Holstein sire (Igale Masc). The identified independent spontaneous splice site variant in COL2A1 most likely caused chondrodysplasia and must have occurred during the early foetal development of the sire. This study provides a first example of a dominant COL2A1 splice site variant as candidate causal mutation of a severe lethal chondrodysplasia phenotype. Germline mosaicism is a relatively frequent mechanism in the origin of genetic disorders and explains the prevalence of a certain fraction of affected offspring. Paternal dominant de novo mutations are a risk in cattle breeding, especially because the ratio of defective offspring may be very high and be associated with significant animal welfare problems.

Evaluation of RET polymorphisms in a six-generation family with G533C RET mutation: specific RET variants may modulate age at onset and clinical presentation

P>Context We previously described a six-generation family with G533C RET mutation and medullary thyroid carcinoma, in the largest family reported do date. Of particular interest, phenotype variability regarding the age of onset and clinical presentation of the disease, was observed. Objective We evaluate whether single SNPs within RET oncogene or haplotype comprising the RET variants (defined by Haploview) could predispose to early development of MTC in this family and influence the clinical manifestation. Design Eight SNPs were selected based on their previous association with the clinical course of hereditary or sporadic MTC, in particular promoting an early onset of disease. The variants were initially tested in 77 G533C-carriers and 100 controls using either PCR-direct sequencing or PCR-RFLP. Association between a SNP or haplotype and age at diagnosis or presence of lymph node metastasis was tested in 34 G533C-carries with MTC. Different bioinformatic tools were used to evaluate the potential effects on RNA splicing. Results An association was found between IVS1-126G > T and age at diagnosis. The variant [IVS8 +82A > G; 85-86 insC] was associated with the presence of lymph node metastases at diagnosis. In silico analysis suggested that this variant may induce abnormal splicing. This in silico analysis predicted that the [IVS8 +82A > G; 85-86 insC] could alter the splicing by disrupting and/or creating exonic splicing enhancer motifs. Conclusions We here identified two RET variants that were associated with phenotype variability in G533C-carriers, which highlights the fact that the modifier effect of a variant might depend on the type of mutation.

Expanding the phenotype and genotype of female GnRH deficiency.

Context: GnRH deficiency is a rare genetic disorder of absent or partial pubertal development. The clinical and genetic characteristics of GnRH-deficient women have not been well-described. Objective: To determine the phenotypic and genotypic spectrum of a large series of GnRH-deficient women. Design, Setting, and Subjects: Retrospective study of 248 females with GnRH deficiency evaluated at an academic medical center between 1980 and 2010. Main Outcome Measures: Clinical presentation, baseline endogenous GnRH secretory activity, and DNA sequence variants in 11 genes associated with GnRH deficiency. Results: Eighty-eight percent had undergone pubarche, 51% had spontaneous thelarche, and 10% had 1-2 menses. Women with spontaneous thelarche were more likely to demonstrate normal pubarche (P = 0.04). In 27% of women, neuroendocrine studies demonstrated evidence of some endogenous GnRH secretory activity. Thirty-six percent (a large excess relative to controls) harbored a rare sequence variant in a gene associated with GnRH deficiency (87% heterozygous and 13% biallelic), with variants in FGFR1 (15%), GNRHR (6.6%), and PROKR2 (6.6%) being most prevalent. One woman had a biallelic variant in the X-linked gene, KAL1, and nine women had heterozygous variants. Conclusions: The clinical presentation of female GnRH deficiency varies from primary amenorrhea and absence of any secondary sexual characteristics to spontaneous breast development and occasional menses. In this cohort, rare sequence variants were present in all of the known genes associated with GnRH deficiency, including the novel identification of GnRH-deficient women with KAL1 variants. The pathogenic mechanism through which KAL1 variants disrupt female reproductive development requires further investigation.

IROme, a New High-Throughput Molecular Tool for the Diagnosis of Inherited Retinal Dystrophies.

The molecular diagnosis of retinal dystrophies is difficult because of the very important number of genes implicated and is rarely helped by genotype-phenotype correlations. This prompted us to develop IROme, a custom designed in solution-based targeted exon capture assay (SeqCap EZ Choice library, Roche NimbleGen) for 60 retinitis pigmentosa-linked genes and three candidate genes (942 exons). Pyrosequencing was performed on a Roche 454 GS Junior benchtop high-throughput sequencing platform. In total, 23 patients affected by retinitis pigmentosa were analyzed. Per patient, 39.6 Mb were generated, and 1111 sequence variants were detected on average, at a median coverage of 17-fold. After data filtering and sequence variant prioritization, disease-causing mutations were identified in ABCA4, CNGB1, GUCY2D, PROM1, PRPF8, PRPF31, PRPH2, RHO, RP2, and TULP1 for twelve patients (55%), ten mutations having never been reported previously. Potential mutations were identified in 5 additional patients, and in only 6 patients no molecular diagnosis could be established (26%). In conclusion, targeted exon capture and next-generation sequencing are a valuable and efficient approach to identify disease-causing sequence variants in retinal dystrophies.

Búsqueda de mutaciones en el gen foxo4 en pacientes con falla ovárica prematura no-sindrómica

FOXO4 constitutes a coherent candidate gene associated with premature ovarian failure (POF) pathogenesis. This study sequenced the coding and exon-flanking regions of this gene in a panel of 116 POF patients and 143 controls of Tunisian origin. In both groups, the IVS2 + 41T > G sequence variant was identified. It is concluded that coding mutations of FOXO4 should not be a common cause of the disease in women from the Tunisian population. However, this study cannot exclude that FOXO4 dysfunctions, originated from open reading frame or promoter sequence variations, might be associated with the pathogenesis of the disease in other ethnical groups.

La variante c.-9C>G del promotor de BMP15 está asociadad a la etiología de la falla ovárica prematura no-sindrómica

La falla ovárica prematura es una enfermedad común que conduce a la infertilidad femenina y cuya etiología no es identificable en más del 50% de los casos, lo cual sugiere un origen genético en la patogénesis de esta enfermedad. La función crucial del gen BMP15 en la biología de la reproducción fue propuesta cuando modelos KO de ratón y mutaciones naturales en ovejas revelaron fenotipos ováricos específicos. Aunque la secuenciación de la región codificante de BMP15 en grandes paneles de pacientes afectadas con Falla Ovárica Prematura (FOP) ha identificado algunas mutaciones, estas variaciones explican una baja proporción de casos. Nosotros hipotetizamos que una variante en la secuencia reguladora (promotor BMP15) podrían estar asociada a la etiología de la FOP no-sindrómica. Con la evidencia de los estudios previos que sugirieron la potencial implicación de la variante de secuencia c.-9C>G del promotor de BMP15 en los fenotipos reproductivos incluyendo FOP, evaluamos si este polimorfismo podría modificar las propiedades de transactivación de un factor de transcripción específico. Empleamos aproximaciones in-silico para predecir potenciales sitios de unión a factores de transcripción (TFBS: Transcription Factor Binding Sites) en la región 5´ de BMP15. El ensayo reportero de luciferasa se usó para determinar la modificación de la transacativación del promotor de BMP15 causada por la variante de c-9C>G. Se demostró que aunque los dos constructos del promotor de BMP15 (BMP15- prom-G and BMP15-prom-C) fueron transactivados por el factor de transcripción PITX1, el constructo BMP15- prom-G aumentó 1.6 veces la actividad transcripcional del factor de transcripción de una manera estadísticamente significativa. Por otro lado, se demostró por primera vez que BMP15 y PITX1 son co-expresados en tejido ovárico de humano y de ratón.

Genetic variation in the oxytocin receptor (OXTR) gene is associated with Asperger Syndrome

Background Autism Spectrum Conditions (ASC) are a group of neurodevelopmental conditions characterized by impairments in communication and social interaction, alongside unusually repetitive behaviors and narrow interests. ASC are highly heritable and have complex patterns of inheritance where multiple genes are involved, alongside environmental and epigenetic factors. Asperger Syndrome (AS) is a subgroup of these conditions, where there is no history of language or cognitive delay. Animal models suggest a role for oxytocin (OXT) and oxytocin receptor (OXTR) genes in social-emotional behaviors, and several studies indicate that the oxytocin/oxytocin receptor system is altered in individuals with ASC. Previous studies have reported associations between genetic variations in the OXTR gene and ASC. Methods The present study tested for an association between nine single nucleotide polymorphisms (SNPs) in the OXTR gene and AS in 530 individuals of Caucasian origin, using SNP association test and haplotype analysis. Results There was a significant association between rs2268493 in OXTR and AS. Multiple haplotypes that include this SNP (rs2268493-rs2254298, rs2268490-rs2268493-rs2254298, rs2268493-rs2254298-rs53576, rs237885-rs2268490-rs2268493-rs2254298, rs2268490-rs2268493-rs2254298-rs53576) were also associated with AS. rs2268493 has been previously associated with ASC and putatively alters several transcription factor-binding sites and regulates chromatin states, either directly or through other variants in linkage disequilibrium (LD). Conclusions This study reports a significant association of the sequence variant rs2268493 in the OXTR gene and associated haplotypes with AS.

Role of the Mitochondrial Mutations, m. 827A > G and the Novel m. 7462C > T, in the Origin of Hearing Loss

Samples from 30 deaf probands exhibiting features suggestive of syndromic mitochondrial deafness or from families with maternal transmission of deafness were selected for investigation of mutations in the mitochondrial genes MT-RNR1 and MT-TS1. Patients with mutation m. 1555A>G had been previously excluded from this sample. In the MT-RNR1 gene, five probands presented the m. 827A>G sequence variant, of uncertain pathogenicity. This change was also detected in 66 subjects of an unaffected control sample of 306 Brazilian individuals from various ethnic backgrounds. Given its high frequency, we consider it unlikely to have a pathogenic role on hereditary deafness. As to the MT-TS1 gene, one proband presented the previously known pathogenic m. 7472insC mutation and three probands presented a novel variant, m. 7462C>T, which was absent from the same control sample of 306 individuals. Because of its absence in control samples and association with a family history of hearing impairment, we suggest it might be a novel pathogenic mutation.

EBNA-1 gene sequences in Brazilian and American patients with Hodgkin's disease

We examined the types of Epstein-Barr virus-associated nuclear antigen-1 (EBNA-1) gene carboxy (C)-terminal mutations occurring in Hodgkin's disease (HD) and reactive tissues from two different geographic regions. Previously reported EBNA-1 C-terminal region amino acid sequence variants, based on the amino acid at codon 487, include Prototype (P)-ala, which is found in the B95.8-derived prototype virus, P-thr, Variant (V)-leu, V-val, and V-pro. Using polymerase chain reaction (PCR) to amplify portions of the EBNA-1 gene, followed by DNA sequencing, we found a single EBNA-1 gene sequence variant in each tissue, whether reactive or neoplastic and whether from Brazil or the United States. Variant EBNA-1 gene sequences were more common in both neoplastic and non-neoplastic tissues from different geographic areas than the so-called prototype sequence. In the 17 Brazilian HD cases, 4 cases had P-thr variants and 13 had V-leu variants. In the six reactive tissues from Brazil, one had a P-ala variant, two had P-thr variants, and three had V-leu variants. In the 12 American HD cases, 2 had P-ala variants, 6 had P-thr variants, and 4 had V-leu variants. The 11 American reactive tissues included 2 P ala variants, 5 P-thr variants, and 4 V-leu variants. In both countries, there were similar variant EBNA-1 sequences present in normal tissues and HD cases. Compared with the P ala and P-thr cases, the V-leu cases were more likely to have the 30-bp latent membrane protein 1 (LMP1) gene deletion (P = 0.0075). In addition, cases of HD with the V-leu were statistically associated with a substitution of asparagine for glutamine at codon 322 of the C-terminal portion of the LMP1 gene. Our results suggest that any variation in EBNA-1 gene sequence is caused by a polymorphism present in pre-existing viral strains in the underlying population, and not a mutation occurring during oncogenesis. (C) 1999 by the American Society of Hematology.

Molecular analysis of carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) as a candidate gene for bovine spongiform encephalopathy susceptibility

Endogenous prion proteins (PrP) play the central role in the pathogenesis of transmissible spongiform encephalopathies. The carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) promotes the conversion of the cellular PrP(C) into the pathogenic PrP(d). Six sequence variants within the CHST8 gene were identified by comparative sequencing and genotyped for a sample of 623 animals comprising bovine spongiform encephalopathy (BSE)-affected and healthy control cows representing German Fleckvieh (German Simmental), German Holstein (Holstein-Friesian) and Brown Swiss. Significant differences in the allele, genotype and haplotype frequencies between BSE-affected and healthy cows indicate an association of sequence variant g.37254017G>T with the development of the disease in Brown Swiss cattle.

Detection of exon deletions within an entire gene (CFTR) by relative quantification on the LightCycler

BACKGROUND: Cystic fibrosis (CF) is associated with at least 1 pathogen point sequence variant on each CFTR allele. Some symptomatic patients, however, have only 1 detectable pathogen sequence variant and carry, on the other allele, a large deletion that is not detected by conventional screening methods. METHODS: For relative quantitative real-time PCR detection of large deletions in the CFTR gene, we designed DNA-specific primers for each exon of the gene and primers for a reference gene (beta2-microglobulin). For PCR we used a LightCycler system (Roche) and calculated the gene-dosage ratio of CFTR to beta2-microglobulin. We tested the method by screening all 27 exons in 3 healthy individuals and 2 patients with only 1 pathogen sequence variant. We then performed specific deletion screenings in 10 CF patients with known large deletions and a blinded analysis in which we screened 24 individuals for large deletions by testing 8 of 27 exons. RESULTS: None of the ratios for control samples were false positive (for deletions or duplications); moreover, for all samples from patients with known large deletions, the calculated ratios for deleted exons were close to 0.5. In addition, the results from the blinded analysis demonstrated that our method can also be used for the screening of single individuals. CONCLUSIONS: The LightCycler assay allows reliable and rapid screening for large deletions in the CFTR gene and detects the copy number of all 27 exons.

Identification and in silico analyses of novel TGFBR1 and TGFBR2 mutations in Marfan syndrome-related disorders

Very recently, heterozygous mutations in the genes encoding transforming growth factor beta receptors I (TGFBR1) and II (TGFBR2) have been reported in Loeys-Dietz aortic aneurysm syndrome (LDS). In addition, dominant TGFBR2 mutations have been identified in Marfan syndrome type 2 (MFS2) and familial thoracic aortic aneurysms and dissections (TAAD). In the past, mutations of these genes were associated with atherosclerosis and several human cancers. Here, we report a total of nine novel and one known heterozygous sequence variants in the TGFBR1 and TGFBR2 genes in nine of 70 unrelated individuals with MFS-like phenotypes who previously tested negative for mutations in the gene encoding the extracellular matrix protein fibrillin-1 (FBN1). To assess the pathogenic impact of these sequence variants, in silico analyses were performed by the PolyPhen, SIFT, and Fold-X algorithms and by means of a 3D homology model of the TGFBR2 kinase domain. Our results showed that in all but one of the patients the pathogenic effect of at least one sequence variant is highly probable (c.722C > T, c.799A > C, and c.1460G > A in TGFBR1 and c.773T > G, c.1106G > T, c.1159G > A, c.1181G > A, and c.1561T > C in TGFBR2). These deleterious alleles occurred de novo or segregated with the disease in the families, indicating a causative association between the sequence variants and clinical phenotypes. Since TGFBR2 mutations found in patients with MFS-related disorders cannot be distinguished from heterozygous TGFBR2 mutations reported in tumor samples, we emphasize the importance of segregation analysis in affected families. In order to be able to find the mutation that is indeed responsible for a MFS-related phenotype, we also propose that genetic testing for sequence alterations in TGFBR1 and TGFBR2 should be complemented by mutation screening of the FBN1 gene.

Keratinocyte transcriptional regulation of the human c-Myc promoter occurs via a novel Lef/Tcf binding element distinct from neoplastic cells

The proto-oncogene c-Myc is involved in early neoplastic transformations. Two consensus Lef/Tcf binding elements (TBE) were found to be prerequisite for transcriptional transactivation by the armadillo proteins beta-catenin and plakoglobin (PG) together with Tcf4 in human neoplastic cells. In epidermal keratinocytes, c-Myc was reported to be repressed by Lef-1 and PG. Using reporter gene assays, here we demonstrate that deletion of the two consensus TBE fails to abrogate transcriptional regulation by Lef-1/PG in wildtype and beta-catenin-/- keratinocytes, while it reduces transcription in pre-neoplastic PG-/- keratinocytes. We identified a TBE sequence variant downstream of the major transcriptional initiation site that binds Lef-1 in vitro and in vivo, and its mutation compromised transcriptional regulation by Lef-1/PG. Collectively, this study demonstrates that the two consensus TBE's reported in neoplastic cells are dispensable for c-Myc regulation in normal keratinocytes, which instead use a novel TBE sequence variant. This unprecedented finding may have important implications for armadillo target genes involved in carcinogenesis.

An equine chromosome 3 inversion is associated with the tobiano spotting pattern in German horse breeds

The tobiano white-spotting pattern is one of several known depigmentation phenotypes in horses and is desired by many horse breeders and owners. The tobiano spotting phenotype is inherited as an autosomal dominant trait. Horses that are heterozygous or homozygous for the tobiano allele (To) are phenotypically indistinguishable. A SNP associated with To had previously been identified in intron 13 of the equine KIT gene and was used for an indirect gene test. The test was useful in several horse breeds. However, genotyping this sequence variant in the Lewitzer horse breed revealed that 14% of horses with the tobiano pattern did not show the polymorphism in intron 13 and consequently the test was not useful to identify putative homozygotes for To within this breed. Speculations were raised that an independent mutation might cause the tobiano spotting pattern in this breed. Recently, the putative causative mutation for To was described as a large chromosomal inversion on equine chromosome 3. One of the inversion breakpoints is approximately 70 kb downstream of the KIT gene and probably disrupts a regulatory element of the KIT gene. We obtained genotypes for the intron 13 SNP and the chromosomal inversion for 204 tobiano spotted horses and 24 control animals of several breeds. The genotyping data confirmed that the chromosomal inversion was perfectly associated with the To allele in all investigated horses. Therefore, the new test is suitable to discriminate heterozygous To/+ and homozygous To/To horses in the investigated breeds.