Identification of eight novel coagulation factor XIII subunit A mutations: implied consequences for structure and function

Severe hereditary coagulation factor XIII deficiency is a rare homozygous bleeding disorder affecting one person in every two million individuals. In contrast, heterozygous factor XIII deficiency is more common, but usually not associated with severe hemorrhage such as intracranial bleeding or hemarthrosis. In most cases, the disease is caused by F13A gene mutations. Causative mutations associated with the F13B gene are rarer.

Brain metabolite composition in relation to cognitive function and dystrophin mutations in boys with Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a hereditary X-linked recessive disorder affecting the synthesis of dystrophin, a protein essential for structural stability in muscle. Dystrophin also occurs in the central nervous system, particularly in the neocortex, hippocampus and cerebellum. Quantitative metabolic analysis by localized (1) H MRS was performed in the cerebellum (12 patients and 15 controls) and a temporo-parietal location (eight patients and 15 controls) in patients with DMD and healthy controls to investigate possible metabolic differences. In addition, the site of individual mutations on the dystrophin gene was analyzed and neuropsychological cognitive functions were examined. Cognitive deficits in the patient group were found in line with earlier investigations, mainly concerning verbal short-term memory, visuo-spatial long-term memory and verbal fluency, but also the full-scale IQ. Causal mutations were identified in all patients with DMD. Quantitative MRS showed consistent choline deficits, in both cerebellar white matter and temporo-parietal cortex, as well as small, but significant, metabolic abnormalities for glutamate and total N-acetyl compounds in the temporo-parietal region. Compartment water analysis did not reveal any abnormalities. In healthy subjects, choline levels were age related in the cerebellum. The choline deficit contrasts with earlier findings in DMD, where a surplus of choline was postulated for the cerebellum. In patients, total N-acetyl compounds in the temporo-parietal region were related to verbal IQ and verbal short-term memory. However, choline, the putative main metabolic abnormality, was not found to be associated with cognitive deficits. Furthermore, in contrast with the cognitive performance, the metabolic brain composition did not depend significantly on whether or not gene mutations concerned the expression of the dystrophin isoform Dp140, leading to the conclusion that the effect of the missing Dp140 isoform on cognitive performance is not mediated through the observed metabolite composition, or is caused by local effects beyond the resolution accessible to MRS investigations.

Neuropsychological impairments and the impact of dystrophin mutations on general cognitive functioning of patients with Duchenne muscular dystrophy

Mutations in the dystrophin gene have long been recognised as a cause of mental retardation. However, for reasons that are unclear, some boys with dystrophin mutations do not show general cognitive deficits. To investigate the relationship between dystrophin mutations and cognition, the general intellectual abilities of a group of 25 boys with genetically confirmed Duchenne muscular dystrophy were evaluated. Furthermore, a subgroup underwent additional detailed neuropsychological assessment. The results showed a mean full scale intelligence quotient (IQ) of 88 (standard deviation 24). Patients performed very poorly on various neuropsychological tests, including arithmetics, digit span tests and verbal fluency. No simple relationship between dystrophin mutations and cognitive functioning could be detected. However, our analysis revealed that patients who lack the dystrophin isoform Dp140 have significantly greater cognitive problems.

Desmosomal gene analysis in arrhythmogenic right ventricular dysplasia/cardiomyopathy: spectrum of mutations and clinical impact in practice

Five desmosomal genes have been recently implicated in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) but the clinical impact of genetics remains poorly understood. We wanted to address the potential impact of genotyping.

Uromodulin is expressed in renal primary cilia and UMOD mutations result in decreased ciliary uromodulin expression

Uromodulin (UMOD) mutations are responsible for three autosomal dominant tubulo-interstitial nephropathies including medullary cystic kidney disease type 2 (MCKD2), familial juvenile hyperuricemic nephropathy and glomerulocystic kidney disease. Symptoms include renal salt wasting, hyperuricemia, gout, hypertension and end-stage renal disease. MCKD is part of the 'nephronophthisis-MCKD complex', a group of cystic kidney diseases. Both disorders have an indistinguishable histology and renal cysts are observed in either. For most genes mutated in cystic kidney disease, their proteins are expressed in the primary cilia/basal body complex. We identified seven novel UMOD mutations and were interested if UMOD protein was expressed in the primary renal cilia of human renal biopsies and if mutant UMOD would show a different expression pattern compared with that seen in control individuals. We demonstrate that UMOD is expressed in the primary cilia of renal tubules, using immunofluorescent studies in human kidney biopsy samples. The number of UMOD-positive primary cilia in UMOD patients is significantly decreased when compared with control samples. Additional immunofluorescence studies confirm ciliary expression of UMOD in cell culture. Ciliary expression of UMOD is also confirmed by electron microscopy. UMOD localization at the mitotic spindle poles and colocalization with other ciliary proteins such as nephrocystin-1 and kinesin family member 3A is demonstrated. Our data add UMOD to the group of proteins expressed in primary cilia, where mutations of the gene lead to cystic kidney disease.

Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizes approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.

Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare form of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.

TILLING for Mutations in Model Plants and Crops

A growing world population, changing climate and limiting fossil fuels will provide new pressures on human production of food, medicine, fuels and feed stock in the twenty-first century. Enhanced crop production promises to ameliorate these pressures. Crops can be bred for increased yields of calories, starch, nutrients, natural medicinal compounds, and other important products. Enhanced resistance to biotic and abiotic stresses can be introduced, toxins removed, and industrial qualities such as fibre strength and biofuel per mass can be increased. Induced and natural mutations provide a powerful method for the generation of heritable enhanced traits. While mainly exploited in forward, phenotype driven, approaches, the rapid accumulation of plant genomic sequence information and hypotheses regarding gene function allows the use of mutations in reverse genetic approaches to identify lesions in specific target genes. Such gene-driven approaches promise to speed up the process of creating novel phenotypes, and can enable the generation of phenotypes unobtainable by traditional forward methods. TILLING (Targeting Induced Local Lesions IN Genome) is a high-throughput and low cost reverse genetic method for the discovery of induced mutations. The method has been modified for the identification of natural nucleotide polymorphisms, a process called Ecotilling. The methods are general and have been applied to many species, including a variety of different crops. In this chapter the current status of the TILLING and Ecotilling methods and provide an overview of progress in applying these methods to different plant species, with a focus on work related to food production for developing nations.

On the traces of XPD: cell cycle matters - untangling the genotype-phenotype relationship of XPD mutations

Abstract Mutations in the human gene coding for XPD lead to segmental progeria - the premature appearance of some of the phenotypes normally associated with aging - which may or may not be accompanied by increased cancer incidence. XPD is required for at least three different critical cellular functions: in addition to participating in the process of nucleotide excision repair (NER), which removes bulky DNA lesions, XPD also regulates transcription as part of the general transcription factor IIH (TFIIH) and controls cell cycle progression through its interaction with CAK, a pivotal activator of cyclin dependent kinases (CDKs). The study of inherited XPD disorders offers the opportunity to gain insights into the coordination of important cellular events and may shed light on the mechanisms that regulate the delicate equilibrium between cell proliferation and functional senescence, which is notably altered during physiological aging and in cancer. The phenotypic manifestations in the different XPD disorders are the sum of disturbances in the vital processes carried out by TFIIH and CAK. In addition, further TFIIH- and CAK-independent cellular activities of XPD may also play a role. This, added to the complex feedback networks that are in place to guarantee the coordination between cell cycle, DNA repair and transcription, complicates the interpretation of clinical observations. While results obtained from patient cell isolates as well as from murine models have been elementary in revealing such complexity, the Drosophila embryo has proven useful to analyze the role of XPD as a cell cycle regulator independently from its other cellular functions. Together with data from the biochemical and structural analysis of XPD and of the TFIIH complex these results combine into a new picture of the XPD activities that provides ground for a better understanding of the patophysiology of XPD diseases and for future development of diagnostic and therapeutic tools.

High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade

Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non-mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRAS(G13D) mutation; one Sézary syndrome and one CD30(+) CTCL harbored a NRAS(Q61K) amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRAS(Q61K) mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRAS(Q61K) mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors.

Phenotypic homogeneity and genotypic variability in a large series of congenital isolated ACTH-deficiency patients with TPIT gene mutations

Congenital isolated ACTH deficiency (IAD) is a rare disease characterized by low plasma ACTH and cortisol levels and preservation of all other pituitary hormones. This condition was poorly defined before we identified TPIT, a T-box transcription factor with a specific role in differentiation of the corticotroph lineage in mice and humans, as its principal molecular cause.

Characterization of novel StAR (steroidogenic acute regulatory protein) mutations causing non-classic lipoid adrenal hyperplasia

Context Steroidogenic acute regulatory protein (StAR) is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH). Objective StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported. Design To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature. Setting Collaboration between the University Children's Hospital Bern, Switzerland, and the CIBERER, Hospital Vall d'Hebron, Autonomous University, Barcelona, Spain. Patients Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age. Results StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (~30%) and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol. Conclusions StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S) seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed.

Metopic and sagittal synostosis in Greig cephalopolysyndactyly syndrome: five cases with intragenic mutations or complete deletions of GLI3

Greig cephalopolysyndactyly syndrome (GCPS) is a multiple congenital malformation characterised by limb and craniofacial anomalies, caused by heterozygous mutation or deletion of GLI3. We report four boys and a girl who were presented with trigonocephaly due to metopic synostosis, in association with pre- and post-axial polydactyly and cutaneous syndactyly of hands and feet. Two cases had additional sagittal synostosis. None had a family history of similar features. In all five children, the diagnosis of GCPS was confirmed by molecular analysis of GLI3 (two had intragenic mutations and three had complete gene deletions detected on array comparative genomic hybridisation), thus highlighting the importance of trigonocephaly or overt metopic or sagittal synostosis as a distinct presenting feature of GCPS. These observations confirm and extend a recently proposed association of intragenic GLI3 mutations with metopic synostosis; moreover, the three individuals with complete deletion of GLI3 were previously considered to have Carpenter syndrome, highlighting an important source of diagnostic confusion.

Collapse of telomere homeostasis in hematopoietic cells caused by heterozygous mutations in telomerase genes

Telomerase activity is readily detectable in extracts from human hematopoietic stem and progenitor cells, but appears unable to maintain telomere length with proliferation in vitro and with age in vivo. We performed a detailed study of the telomere length by flow FISH analysis in leukocytes from 835 healthy individuals and 60 individuals with reduced telomerase activity. Healthy individuals showed a broad range in average telomere length in granulocytes and lymphocytes at any given age. The average telomere length declined with age at a rate that differed between age-specific breakpoints and between cell types. Gender differences between leukocyte telomere lengths were observed for all cell subsets studied; interestingly, this trend could already be detected at birth. Heterozygous carriers for mutations in either the telomerase reverse transcriptase (hTERT) or the telomerase RNA template (hTERC) gene displayed striking and comparable telomere length deficits. Further, non-carrier relatives of such heterozygous individuals had somewhat shorter leukocyte telomere lengths than expected; this difference was most profound for granulocytes. Failure to maintain telomere homeostasis as a result of partial telomerase deficiency is thought to trigger cell senescence or cell death, eventually causing tissue failure syndromes. Our data are consistent with these statements and suggest that the likelihood of similar processes occurring in normal individuals increases with age. Our work highlights the essential role of telomerase in the hematopoietic system and supports the notion that telomerase levels in hematopoietic cells, while limiting and unable to prevent overall telomere shortening, are nevertheless crucial to maintain telomere homeostasis with age.