Hereditary truncating mutations of DNA repair and other genes in BRCA1/BRCA2/PALB2-negatively tested breast cancer patients.

Hereditary breast cancer comprises a minor but clinically meaningful breast cancer (BC) subgroup. Mutations in the major BC-susceptibility genes are important prognostic and predictive markers; however, their carriers represent only 25% of high-risk BC patients. To further characterize variants influencing BC risk, we performed SOLiD sequencing of 581 genes in 325 BC patients (negatively tested in previous BRCA1/BRCA2/PALB2 analyses). In 105 (32%) patients, we identified and confirmed 127 truncating variants (89 unique; nonsense, frameshift indels, and splice site), 19 patients harbored more than one truncation. Forty-six (36 unique) truncating variants in 25 DNA repair genes were found in 41 (12%) patients, including 16 variants in the Fanconi anemia (FA) genes. The most frequent variant in FA genes was c.1096_1099dupATTA in FANCL that also show a borderline association with increased BC risk in subsequent analysis of enlarged groups of BC patients and controls. Another 81 (53 unique) truncating variants were identified in 48 non-DNA repair genes in 74 patients (23%) including 16 patients carrying variants in genes coding proteins of estrogen metabolism/signaling. Our results highlight the importance of mutations in the FA genes' family, and indicate that estrogen metabolism genes may reveal a novel candidate genetic component for BC susceptibility.

Clinical and molecular characterization of a family with a dominant renin gene mutation and response to treatment with fludrocortisone.

BACKGROUND: A family was identified with autosomal dominant inheritance of anemia, polyuria, hyperuricemia, and chronic kidney disease. Mutational analysis revealed a novel heterozygous mutation c.58T > C resulting in the amino acid substitution of cysteine for arginine in the preprorenin signal sequence (p.cys20Arg) occurring in all affected members. METHODS: Effects of the identified mutation were characterized using in vitro and in vivo studies. Affected individuals were clinically characterized before and after administration of fludrocortisone. RESULTS: The mutation affects endoplasmic reticulum co-translational translocation and posttranslational processing, resulting in massive accumulation of non-glycosylated preprorenin in the cytoplasm. This affects expression of intra-renal RAS components and leads to ultrastructural damage of the kidney. Affected individuals suffered from anemia, hyperuricemia, decreased urinary concentrating ability, and progressive chronic kidney disease. Treatment with fludrocortisone in an affected 10-year-old child resulted in an increase in blood pressure and estimated glomerular filtration rate. CONCLUSIONS: A novel REN gene mutation resulted in an alteration in the amino acid sequence of the renin signal sequence and caused childhood anemia, polyuria, and kidney disease. Treatment with fludrocortisone improved renal function in an affected child. Nephrologists should consider REN mutational analysis in families with autosomal dominant inheritance of chronic kidney disease, especially if they suffer from anemia, hyperuricemia, and polyuria in childhood.

Large copy-number variations in patients with statin-associated myopathy affecting statin myopathy-related loci.

Some patients are susceptible to statin-associated myopathy (SAM) either because of genetic variations affecting statin uptake and metabolism, or because they predispose their carriers to muscular diseases. Among the frequent variants examined using the genome-wide association study approach, SLCO1B1 c.521T>C represents the only validated predictor of SAM in patients treated with high-dose simvastatin. Our aim was to ascertain the overall contribution of large copy-number variations (CNVs) to SAM diagnosed in 86 patients. CNVs were detected by whole genome genotyping using Illumina HumanOmni2.5 Exome BeadChips. Exome sequence data were used for validation of CNVs in SAM-related loci. In addition, we performed a specific search for CNVs in the SLCO1B region detected recently in Rotor syndrome subjects. Rare deletions possibly contributing to genetic predisposition to SAM were found in two patients: one removed EYS associated previously with SAM, the other was present in LARGE associated with congenital muscular dystrophy. Another two patients carried deletions in CYP2C19, which may predispose to clopidogrel-statin interactions. We found no common large CNVs potentially associated with SAM and no CNVs in the SLCO1B locus. Our findings suggest that large CNVs do not play a substantial role in the etiology of SAM.

Mutations in PNPLA6 are linked to photoreceptor degeneration and various forms of childhood blindness.

Blindness due to retinal degeneration affects millions of people worldwide, but many disease-causing mutations remain unknown. PNPLA6 encodes the patatin-like phospholipase domain containing protein 6, also known as neuropathy target esterase (NTE), which is the target of toxic organophosphates that induce human paralysis due to severe axonopathy of large neurons. Mutations in PNPLA6 also cause human spastic paraplegia characterized by motor neuron degeneration. Here we identify PNPLA6 mutations in childhood blindness in seven families with retinal degeneration, including Leber congenital amaurosis and Oliver McFarlane syndrome. PNPLA6 localizes mostly at the inner segment plasma membrane in photoreceptors and mutations in Drosophila PNPLA6 lead to photoreceptor cell death. We also report that lysophosphatidylcholine and lysophosphatidic acid levels are elevated in mutant Drosophila. These findings show a role for PNPLA6 in photoreceptor survival and identify phospholipid metabolism as a potential therapeutic target for some forms of blindness.

A case of type I Gaucher disease with cardiopulmonary amyloidosis and chitotriosidase deficiency.

Severe cardiopulmonary amyloidosis developed several months after a total splenectomy in a patient with type 1 Gaucher disease and led within a year to his death at 48 years of age. The autopsy findings were dominated by extensive pulmonary and cardiac amyloid infiltration. No Gaucher cells were found in the lungs. Aside from a glucocerebrosidase deficiency the patient was also deficient in chitotriosidase, an enzyme whose activity is usually greatly increased in the serum of Gaucher patients. Analysis of mutations in the glucocerebrosidase gene revealed heterozygosity for N370S and D409H mutations. The normal amount of glucocerebrosidase was found in the spleen by Western blotting. We suggest that amyloidosis should be considered in the differential diagnosis of severe cardiopulmonary disease in Gaucher patients.

Interaction of a bZip oligopeptide model with oligodeoxyribonucleotides modelling DNA binding sites. The effect of flanking sequences.

A leucine zipper (bZip) binding peptide BP1 was constructed based on the DNA binding sequence of the GCN4 protein, slightly modified to make it more similar to the sequence of other bZip proteins (Jun) with related DNA binding specificity. Self-complementary DNA hexadecanucleotides containing ATF/CRE, AP-1 and C/EPB target sites were used to study peptide-DNA complex formation. Conformation changes in both components that occur on complex formation were studied by circular dichroism (CD) spectroscopy. The results show that the amount of alpha-helix formed in the peptide strongly depends not only on the target site present, but also on the type of the sequence flanking the ATF/CRE target site. Highest amount of the alpha-helix induced in the peptide was observed when homopurine homopyrimidine flanking sequences were present, whereas the presence of alternating sequences, especially of the CA/TG type, showed considerably lower effects. The change in DNA conformation on complex formation was generally small, but also depended on the type of the flanking sequence. It appears that the sequences flanking the target site can considerably modify the ability of the target sequence to bind specifically the bZip peptide, probably by slightly varying the overall DNA conformation.

Cells of porcine epidermis and corneal epithelium are not recognized by human natural anti-alpha-galactoside IgG.

Human natural antibodies against Galalphal,3Gal-R are mainly responsible for hyperacute rejection of xenografts transplanted to the human host. In addition to the anti-alpha-Gal activity, human serum also contains anti-beta-Gal IgG fractions. Employing biotinylated IgG subfractions with anti-alpha- and anti-beta-Gal activity purified from human natural IgG, we have studied expression of reactive epitopes in porcine and human skin, porcine cultured keratinocytes and porcine and human cornea, porcine liver and human lacrimal gland, tear fluid and capillaries. No reactivity of porcine and human epidermis as well as anterior corneal epithelium was observed for human anti-alpha-Gal IgG. Serving as positive control, porcine capillaries gave the expected signal with the anti-alpha-Gal antibody. The anti-beta-Gal subfraction recognized cell nuclei in the epidermis of both these species. The pig liver cells interacted with antibodies against alpha- and beta-galactosides like cells of the human lacrimal gland. alpha-galactoside-reactive glycoproteins were also detected in the human tear fluid. The carbohydrate specificity of the reaction was ascertained by using melibiose as competitive sugar for alpha-galactoside-mediated binding. These results reveal the presentation of Galalpha1,3Gal in epithelial cells of human lacrimal gland, its biosynthetic origin being unclear. With respect to a potential clinical perspective, the given results facilitate consideration of the use of porcine epidermal cells in engineering of non-permanent wound covers to improve treatment.

[Enzyme therapy in children with severe forms of Gaucher's disease]. / Enzymová terapie u detí s tezkou formou Gaucherovy nemoci.

The enzyme therapy with Ceredase in patients with Gaucher's disease is at present probably the most expensive treatment in the whole world. One-year treatment of an adult patient with Gaucher's disease costs more than 7 million crowns. Indications for treatment in individual patients as well as financial provisions are so far problematic in the Czech Republic. From a total of 28 patients of varying age with Gaucher's disease diagnosed by the authors Ceredase was administered to two boys with a severe course of the disease. Within one year of treatment the health status of both children improved, growth became normal, the spleen diminished in size by 20-35%, haematological manifestations of hypersplenism are receding, there was a 32-46% decline of the activity of serum chitotriosidase and biochemical parameters of the disease improved.

Alterations of uromodulin biology: a common denominator of the genetically heterogeneous FJHN/MCKD syndrome.

Autosomal dominant hyperuricemia, gout, renal cysts, and progressive renal insufficiency are hallmarks of a disease complex comprising familial juvenile hyperuricemic nephropathy and medullary cystic kidney diseases type 1 and type 2. In some families the disease is associated with mutations of the gene coding for uromodulin, but the link between the genetic heterogeneity and mechanism(s) leading to the common phenotype symptoms is not clear. In 19 families, we investigated relevant biochemical parameters, performed linkage analysis to known disease loci, sequenced uromodulin gene, expressed and characterized mutant uromodulin proteins, and performed immunohistochemical and electronoptical investigation in kidney tissues. We proved genetic heterogeneity of the disease. Uromodulin mutations were identified in six families. Expressed, mutant proteins showed distinct glycosylation patterns, impaired intracellular trafficking, and decreased ability to be exposed on the plasma membrane, which corresponded with the observations in the patient's kidney tissue. We found a reduction in urinary uromodulin excretion as a common feature shared by almost all of the families. This was associated with case-specific differences in the uromodulin immunohistochemical staining patterns in kidney. Our results suggest that various genetic defects interfere with uromodulin biology, which could lead to the development of the common disease phenotype. 'Uromodulin-associated kidney diseases' may be thus a more appropriate term for this syndrome.

Analysis of the beta-glucocerebrosidase gene in Czech and Slovak Gaucher patients: mutation profile and description of six novel mutant alleles.

The aim of this study was to characterize the spectrum of 13-glucocerebrosidase gene mutations in Czech and Slovak Gaucher patients and to study genotype/phenotype associations. We have analyzed fifty-eight chromosomes from twenty-six type I, two type 2, and one type 3 13-glucocerebrosidase deficient subjects by direct sequencing of PCR products. Fifty-eight mutant alleles were identified. Seventy-eight percent of mutant alleles carried common mutations (N370S 28/58, L444P 11/58, recNciI 5/58, and IVS2(+1)A 1/58), the remaining twenty-two percent carried rare and private mutations (1263del55, l326insT, S196P, rec(g4889-6506), 2O3delC, G202E, F216Y, R257X, R12OW, R359Q, S1O7L, L444P + V460V, and D409H + T369M). Six of these alleles have not been previously described (rec(g4889-6506), 1326insT, SI96P. G202E, D409H + T369M, and L444P + V460V). The most common genotypes were N370S/L444P (8/29). N370S/recNciI (5/29), and N370S/N370S (2/29). The spectrum of the mutations is characteristic for a Caucasian (non-Jewish) population, with N370S, L444P and recNciI being the most prevalent mutations. The absence of the mutation 84insG that is frequently associated with severe bone disease may have contributed to the low incidence of severe bone disease in Czech and Slovak Gaucher subjects.