Germline mutation analysis of Tpit in Poodle dogs with ACTH-dependent hyperadrenocorticism

There is a high incidence of pituitary-dependent hyperadrenocorticism (PDH) in Poodle dogs, with family members being affected by the disease, suggesting a genetic involvement. Tpit is an obligate transcription factor for the expression of pro-opiomelanocortingene and for corticotroph terminal differentiation. The aim of the present study was to screen the Tpit gene for germline mutations in Poodles with PDH. Fifty Poodle dogs (33 female, 8.71 +/- 2.8 years) with PDH and 50 healthy Poodle dogs (32 females, 9.4241 2.8 years) were studied. Genomic DNA was isolated from peripheral blood, amplified by PCR and submitted to automatic sequence. No mutation in the coding region of Tpit was found, whereas the new single nucleotide polymorphism p.S343G, in heterozygous state, was found in the same frequency in both PDH and control groups. We concluded that Tpit gain-of-function mutations are not involved in the etiology of PDH in Poodle dogs.

Mutation analysis of NPHP6/CEP290 in patients with Joubert syndrome and Senior-Løken syndrome

BACKGROUND: Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease that constitutes the most common genetic cause of renal failure in the first three decades of life. Using positional cloning, six genes (NPHP1-6) have been identified as mutated in NPHP. In Joubert syndrome (JBTS), NPHP may be associated with cerebellar vermis aplasia/hypoplasia, retinal degeneration and mental retardation. In Senior-Løken syndrome (SLSN), NPHP is associated with retinal degeneration. Recently, mutations in NPHP6/CEP290 were identified as a new cause of JBTS. METHODS: Mutational analysis was performed on a worldwide cohort of 75 families with SLSN, 99 families with JBTS and 21 families with isolated nephronophthisis. RESULTS: Six novel and six known truncating mutations, one known missense mutation and one novel 3 bp pair in-frame deletion were identified in a total of seven families with JBTS, two families with SLSN and one family with isolated NPHP.

Mutation analysis of the muscarinic cholinergic receptor genes in isolated growth hormone deficiency type IB

BACKGROUND: Isolated GH deficiency (IGHD) is familial in 5-30% of patients. The most frequent form (IGHD-IB) has autosomal recessive inheritance, and it is known that it can be caused by mutations in the GHRH receptor (GHRHR) gene or in the GH gene. However, most forms of IGHD-IB have an unknown genetic cause. In normal subjects, muscarinic cholinergic stimulation causes an increase in pituitary GH release, whereas its blockade has the opposite effect, suggesting that a muscarinic acetylcholine receptor (mAchR) is involved in stimulating GH secretion. Five types of mAchR (M(1)-M(5)) exist. A transgenic mouse in which the function of the M(3) receptor was selectively ablated in the central nervous system has isolated GH deficiency similar to animals with defective GHRH or GHRHR gene. OBJECTIVE: We hypothesized that mAchR mutations may cause a subset of familial IGHD. PATIENTS/METHODS: After confirming the expression of M(1)-M(5) receptor mRNA in human hypothalamus, we analyzed the index cases of 39 families with IGHD-IB for mutations in the genes encoding for the five receptors. Coding sequences for each of the five mAchRs were subjected to direct sequencing. RESULTS: In one family, an affected member was homozygous for a M(3) change in codon 65 that replaces valine with isoleucine (V65I). The V65I receptor was expressed in CHO cells where it had normal ability to transmit methacholine signaling. CONCLUSION: mAchR mutations are absent or rare (less than 2.6%) in familial IGHD type IB.

Mutation analysis of the PVRL1 gene in caucasians with nonsyndromic cleft lip/palate.

Nonsyndromic cleft lip with or without cleft palate (nsCL/P, MIM 119530) is perhaps the most common major birth defect. Homozygous PVRL1 loss-of-function mutations result in an autosomal recessive CL/P syndrome, CLPED1, and a PVRL1 nonsense mutation is associated with sporadic nsCL/P in Northern Venezuela. To address the more general role of PVRL1 variation in risk of nsCL/P, we carried out mutation analysis of PVRL1 in North American and Australian nsCL/P cases and population-matched controls. We identified a total of 15 variants, 5 of which were seen in both populations and 1 of which, an in-frame insertion at Glu442, was more frequent in patients than in controls in both populations, though the difference was not statistically significant. Another variant, which is specific to the PVRL1 beta (HIgR) isoform, S447L, was marginally associated with nsCL/P in North American Caucasian patients, but not in Australian patients, and overall variants that affect the beta-isoform were significantly more frequent among North American patients. One Australian patient had a splice junction mutation of PVRL1. Our results suggest that PVRL1 may play a minor role in susceptibility to the occurrence of nsCL/P in some Caucasian populations, and that variation involving the beta (HIgR) isoform might have particular importance for risk of orofacial clefts. Nevertheless, these results underscore the need for studies that involve very large numbers when assessing the possible role of rare variants in risk of complex traits such as nsCL/P.

Fine-mapping and mutation analysis of TRPM1: a candidate gene for leopard complex (LP) spotting and congenital stationary night blindness in horses.

Leopard Complex spotting occurs in several breeds of horses and is caused by an incompletely dominant allele (LP). Homozygosity for LP is also associated with congenital stationary night blindness (CSNB) in Appaloosa horses. Previously, LP was mapped to a 6 cm region on ECA1 containing the candidate gene TRPM1 (Transient Receptor Potential Cation Channel, Subfamily M, Member 1) and decreased expression of this gene, measured by qRT-PCR, was identified as the likely cause of both spotting and ocular phenotypes. This study describes investigations for a mutation causing or associated with the Leopard Complex and CSNB phenotype in horses. Re-sequencing of the gene and associated splice sites within the 105 624 bp genomic region of TRPM1 led to the discovery of 18 SNPs. Most of the SNPs did not have a predictive value for the presence of LP. However, one SNP (ECA1:108,249,293 C>T) found within intron 11 had a strong (P < 0.0005), but not complete, association with LP and CSNB and thus is a good marker but unlikely to be causative. To further localize the association, 70 SNPs spanning over two Mb including the TRPM1 gene were genotyped in 192 horses from three different breeds segregating for LP. A single 173 kb haplotype associated with LP and CSNB (ECA1: 108,197,355- 108,370,150) was identified. Illumina sequencing of 300 kb surrounding this haplotype revealed 57 SNP variants. Based on their localization within expressed sequences or regions of high sequence conservation across mammals, six of these SNPs were considered to be the most likely candidate mutations. While the precise function of TRPM1 remains to be elucidated, this work solidifies its functional role in both pigmentation and night vision. Further, this work has identified several potential regulatory elements of the TRPM1 gene that should be investigated further in this and other species.

Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly

At least 11 complementation groups (CGs) have been identified for the peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, for which seven pathogenic genes have been elucidated. We have isolated a human PEX19 cDNA (HsPEX19) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary cell line, ZP119, defective in import of both matrix and membrane proteins. This cDNA encodes a hydrophilic protein (Pex19p) comprising 299 amino acids, with a prenylation motif, CAAX box, at the C terminus. Farnesylated Pex19p is partly, if not all, anchored in the peroxisomal membrane, exposing its N-terminal part to the cytosol. A stable transformant of ZP119 with HsPEX19 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX19 expression also restored peroxisomal protein import in fibroblasts from a patient (PBDJ-01) with Zellweger syndrome of CG-J. This patient (PBDJ-01) possessed a homozygous, inactivating mutation: a 1-base insertion, A764, in a codon for Met255, resulted in a frameshift, inducing a 24-aa sequence entirely distinct from normal Pex19p. These results demonstrate that PEX19 is the causative gene for CG-J PBD and suggest that the C-terminal part, including the CAAX homology box, is required for the biological function of Pex19p. Moreover, Pex19p is apparently involved at the initial stage in peroxisome membrane assembly, before the import of matrix protein.

Mutation analysis to verify feature matrices for isolating errors in simulation models

Software simulation models are computer programs that need to be verified and debugged like any other software. In previous work, a method for error isolation in simulation models has been proposed. The method relies on a set of feature matrices that can be used to determine which part of the model implementation is responsible for deviations in the output of the model. Currrently these feature matrices have to be generated by hand from the model implementation, which is a tedious and error-prone task. In this paper, a method based on mutation analysis, as well as prototype tool support for the verification of the manually generated feature matrices is presented. The application of the method and tool to a model for wastewater treatment shows that the feature matrices can be verified effectively using a minimal number of mutants.

ERIC recommendations on TP53 mutation analysis in chronic lymphocytic leukemia.

Recent evidence suggests that - in addition to 17p deletion - TP53 mutation is an independent prognostic factor in chronic lymphocytic leukemia (CLL). Data from retrospective analyses and prospective clinical trials show that ∼5% of untreated CLL patients with treatment indication have a TP53 mutation in the absence of 17p deletion. These patients have a poor response and reduced progression-free survival and overall survival with standard treatment approaches. These data suggest that TP53 mutation testing warrants integration into current diagnostic work up of patients with CLL. There are a number of assays to detect TP53 mutations, which have respective advantages and shortcomings. Direct Sanger sequencing of exons 4-9 can be recommended as a suitable test to identify TP53 mutations for centers with limited experience with alternative screening methods. Recommendations are provided on standard operating procedures, quality control, reporting and interpretation. Patients with treatment indications should be investigated for TP53 mutations in addition to the work-up recommended by the International workshop on CLL guidelines. Patients with TP53 mutation may be considered for allogeneic stem cell transplantation in first remission. Alemtuzumab-based regimens can yield a substantial proportion of complete responses, although of short duration. Ideally, patients should be treated within clinical trials exploring new therapeutic agents.

Refining the diagnosis and EGFR status of non-small cell lung carcinoma in biopsy and cytologic material, using a panel of mucin staining, TTF-1, cytokeratin 5/6, and P63, and EGFR mutation analysis.

INTRODUCTION: The dichotomization of non-small cell carcinoma (NSCLC) subtype into squamous (SQCC) and adenocarcinoma (ADC) has become important in recent years and is increasingly required with regard to management. The aim of this study was to determine the utility of a panel of commercially available antibodies in refining the diagnosis on small biopsies and also to determine whether cytologic material is suitable for somatic EGFR genotyping in a prospectively analyzed series of patients undergoing investigation for suspected lung cancer. METHODS: Thirty-two consecutive cases of NSCLC were first tested using a panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, 34betaE12, and a D-PAS stain for mucin, to determine their value in refining diagnosis of NSCLC. After this test phase, two further pathologists independently reviewed the cases using a refined panel that excluded 34betaE12 because of its low specificity for SQCC, and refinement of diagnosis and concordance were assessed. Ten cases of ADC, including eight derived from cytologic samples, were sent for EGFR mutation analysis. RESULTS: There was refinement of diagnosis in 65% of cases of NSCLC to either SQCC or ADC in the test phase. This included 10 of 13 cases where cell pellets had been prepared from transbronchial needle aspirates. Validation by two further pathologists with varying expertise in lung pathology confirmed increased refinement and concordance of diagnosis. All samples were adequate for analysis, and they all showed a wild-type EGFR genotype. CONCLUSION: A panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, and a D-PAS stain for mucin increases diagnostic accuracy and agreement between pathologists when faced with refining a diagnosis of NSCLC to SQCC or ADC. These small samples, even cell pellets derived from transbronchial needle aspirates, seem to be adequate for EGFR mutation analysis.

Haplotype analysis suggest common founders in carriers of the recurrent BRCA2 mutation, 3398delAAAAG, in French Canadian hereditary breast and/ovarian cancer families

Affiliation: Faculté de médicine, Université de Montréal