Mutations in the gamma 2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is an autosomal recessive disorder characterized by blister formation within the dermal-epidermal basement membrane. Genes for the lamina lucida protein, kalinin/laminin 5, have been proposed as candidates for some forms of JEB, based on immunofluorescence analysis recognizing kalinin epitopes. We studied the cDNA of laminin gamma 2 chain for mutations in JEB using heteroduplex analysis. One patient showed a homozygous splice site mutation while another was heterozygous for a deletion-insertion, resulting in a premature termination codon in one allele. Our data implicate mutations in the laminin gamma 2 chain gene (LAMC2) in some forms of JEB.

Herlitz's junctional epidermolysis bullosa is linked to mutations in the gene (LAMC2) for the gamma 2 subunit of nicein/kalinin (LAMININ-5).

We have linked Herlitz's junctional epidermolysis bullosa (H-JEB) to the gene (LAMC2) encoding the gamma 2 subunit of nicein/kalinin, an isolaminin (laminin-5) expressed by basal keratinocytes. In four H-JEB kindreds, a maximum two-point lod score of 5.33 at theta = 0 was observed between a microsatellite near LAMC2 at 1q25-31 and the disease. In one family, a homozygous point mutation leading to a premature stop codon (CGA to TGA) was identified in exon 3 of the gene. The segregation of the mutated allele implicates the mutation in the pathology of the disorder and corroborates the linkage results.

Integrin beta 4 mutations associated with junctional epidermolysis bullosa with pyloric atresia.

Pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB), is a rare inherited disorder characterized by pyloric stenosis and blistering of the skin as primary manifestations. We demonstrate that in one PA-JEB patient the disease resulted from two distinct mutations in the beta 4 integrin gene alleles. The paternal mutation consists of a one base pair deletion causing a shift in the open reading frame, and a downstream premature termination codon. The maternal mutation occurs in a donor splice site, and results in in-frame exon skipping involving the cytoplasmic domain of the polypeptide. Our results implicate mutations in the beta 4 integrin gene in some forms of PA-JEB.

Plectin deficiency results in muscular dystrophy with epidermolysis bullosa.

We report that mutation in the gene for plectin, a cytoskeleton-membrane anchorage protein, is a cause of autosomal recessive muscular dystrophy associated with skin blistering (epidermolysis bullosa simplex). The evidence comes from absence of plectin by antibody staining in affected individuals from four families, supportive genetic analysis (localization of the human plectin gene to chromosome 8q24.13-qter and evidence for disease segregation with markers in this region) and finally the identification of a homozygous frameshift mutation detected in plectin cDNA. Absence of the large multifunctional cytoskeleton protein plectin can simultaneously account for structural failure in both muscle and skin.

Prospective relationships of ADHD symptoms with developing substance use in a population-derived sample.

BACKGROUND: Clinically ascertained reports suggest that boys and girls with attention deficit hyperactivity disorder (ADHD) may differ from each other in their vulnerability to substance use problems. METHOD: A total of 1545 Finnish adolescents were assessed for DSM-IV-based ADHD symptoms by their parents and classroom teachers using standardized rating scales at age 11-12 years. At age 14, substance use disorders and psychiatric co-morbidity were assessed with the Semi-Structured Assessment for the Genetics of Alcoholism, providing DSM-III-R/DSM-IV diagnoses for Axis I disorders. At age 17.5, substance use was assessed by multi-item questionnaire. RESULTS: Although baseline ADHD symptoms were less common among females, they were more predictive of adverse substance use outcomes once conduct disorder and previous substance use were controlled for. Only in females were baseline ADHD symptoms significant predictors of alcohol abuse and dependence and illicit drug use at age 14. At the age of 17.5, parents' reports of inattentiveness and hyperactivity were significant predictors for frequent alcohol use in both sexes, but they were more predictive of frequent alcohol and illicit drug use in girls. Impulsivity in teachers' ratings predicted frequent alcohol use and illicit drug use in boys. Parental reports of inattentiveness in their 11-/12-year-old daughters were a consistent predictor for illicit drug use across adolescence. CONCLUSIONS: Inattentiveness and hyperactivity may be more predictive of alcohol use disorders and maladaptive patterns of alcohol and illicit drug use among girls than boys. The importance of these behavioural symptoms should be assessed further in the community, as they could jeopardize adolescents' successful transitioning into adult roles.

Ketone body 3-hydroxybutyrate as a biomarker of aggression.

Human aggression is a complex behaviour, the biological underpinnings of which remain poorly known. To gain insights into aggression biology, we studied relationships with aggression of 11 low-molecular-weight metabolites (amino acids, ketone bodies), processed using 1H nuclear magnetic resonance spectroscopy. We used a discovery sample of young adults and an independent adult replication sample. We studied 725 young adults from a population-based Finnish twin cohort born 1983-1987, with aggression levels rated in adolescence (ages 12, 14, 17) by multiple raters and blood plasma samples at age 22. Linear regression models specified metabolites as the response variable and aggression ratings as predictor variables, and included several potential confounders. All metabolites showed low correlations with aggression, with only one-3-hydroxybutyrate, a ketone body produced during fasting-showing significant (negative) associations with aggression. Effect sizes for different raters were generally similar in magnitude, while teacher-rated (age 12) and self-rated (age 14) aggression were both significant predictors of 3-hydroxybutyrate in multi-rater models. In an independent replication sample of 960 adults from the Netherlands Twin Register, higher aggression (self-rated) was also related to lower levels of 3-hydroxybutyrate. These exploratory epidemiologic results warrant further studies on the role of ketone metabolism in aggression.

Link between plasma ceramides, inflammation and insulin resistance: association with serum IL-6 concentration in patients with coronary heart disease.

AIMS/HYPOTHESIS: Ceramides and IL-6 have a role in immune-inflammatory responses and cardiovascular diseases, and are suggested to be involved in insulin and glucose metabolism. We sought to assess the associations of circulating levels of IL-6, TNF-alpha and high-sensitivity C reactive protein (hsCRP), which are inflammatory markers related to insulin resistance (IR), with the plasma lipid metabolites ceramides and diacylglycerols (DAG) in patients with CHD. METHODS: Cross-sectional analyses were carried out on data from 33 patients with CHD. Serum levels of the inflammatory markers and plasma lipid metabolites (lipidomics approach performed by ultra-performance liquid chromatography coupled to electrospray ionisation MS) were measured at the same time point as insulin resistance (IR) (HOMA-IR index). RESULTS: Serum circulating levels of IL-6 were strongly correlated with plasma ceramide concentrations (r = 0.59, p < 0.001). Adjustments for serum TNF-alpha or hsCRP levels, smoking, BMI, age, sex or HOMA-IR did not change the results (p < 0.001). After adjustments for the effect of serum inflammatory markers (TNF-alpha or hsCRP), HOMA-IR and BMI the correlation between plasma DAG and serum IL-6 (r = 0.33) was also significant (p < 0.03). In a linear regression model, circulating levels of both ceramides and TNF-alpha had a significant independent influence on circulating levels of IL-6, altogether accounting for 41% of its variation (p < 0.001). CONCLUSIONS/INTERPRETATION: Our results strongly suggest that the link between ceramides, IR and inflammation is related to the inflammatory marker IL-6. Ceramides may contribute to the induction of inflammation involved in IR states that frequently coexist with CHD.

Genetic and environmental effects on body mass index during adolescence: a prospective study among Finnish twins.

OBJECTIVE: To study genetic and environmental factors affecting body mass index (BMI) and BMI phenotypic correlations across adolescence. DESIGN: Prospective, population-based, twin cohort study. PARTICIPANTS AND METHODS: We used twin modeling in 2413 monozygotic and same-sex and opposite-sex dizygotic Finnish twin pairs born in 1983-1987 and assessed using self-report questionnaires at 11-12, 14 and 17 years of age. RESULTS: Heritability of BMI was estimated to be 0.58-0.69 among 11-12- and 14-year-old boys and girls, 0.83 among 17-year-old boys and 0.74 among 17-year-old girls. Common environmental effects shared by siblings were 0.15-0.24 among 11-12- and 14-year-old boys and girls but no longer discernible at 17 years of age. Unique environmental effects were 0.15-0.23. Additive genetic factors explained 90-96% of the BMI phenotypic correlations across adolescence, whereas unique environmental factors explained the rest. Common environment had no effect on BMI phenotypic correlations. CONCLUSIONS: The genetic contribution to BMI is strong during adolescence, and it mainly explains BMI phenotypic correlations across adolescence. Common environmental factors have an effect on BMI during early adolescence, but that effect disappears by late adolescence.

Association of sequence variations in the gene encoding insulin-like growth factor binding protein 5 with adiponectin.

BACKGROUND: Insulin-like growth factor binding protein 5 (IGFBP5) binds to IGF and thus modulates IGF signaling pathway. We have shown earlier that the IGFBP5 gene was downregulated in the adipose tissue after 12-week carbohydrate diet with low insulinemic response. OBJECTIVE: The aim was to examine the putative contribution of genetic variation of the IGFBP5 gene to the characteristics of metabolic syndrome and incidence of type 2 diabetes (T2DM) in the Finnish Diabetes Prevention Study (DPS). METHODS: DPS is a longitudinal study where 522 subjects with impaired glucose tolerance were randomized to either lifestyle intervention group or control group. DNA was available from 507 subjects (mean body mass index (BMI) 31.2+/-4.5 kg/m(2), age 55+/-7 years). The eight single-nucleotide polymorphisms (SNPs) were selected from HapMap database and genotyped by Taqman allelic discrimination protocol. The main results were confirmed in a larger cross-sectional study population (METSIM). In addition, the gene expression of IGFBP5 was studied in two previously published study populations (FUNGENUT and GENOBIN) of 124 subjects with insulin resistance (BMI 32.2+/-3.5 kg/m(2), age 57.7+/-7.4 years). RESULTS: Three out of eight IGFBP5 markers (rs9341234, rs3276 and rs11575134) were significantly associated with circulating adiponectin concentrations in men. Furthermore, mRNA expression studies of subcutaneous adipose tissue showed that mRNA concentrations of IGFBP5 correlated with adiponectin concentrations in all subjects and in women. None of the IGFBP5 SNPs were associated with T2DM. CONCLUSIONS: Our findings show that IGFBP5 has a gender-specific association with adiponectin, which may modulate the development of metabolic syndrome.

Targeted disruption of the pemphigus vulgaris antigen (desmoglein 3) gene in mice causes loss of keratinocyte cell adhesion with a phenotype similar to pemphigus vulgaris.

In patients with pemphigus vulgaris (PV), autoantibodies against desmoglein 3 (Dsg3) cause loss of cell-cell adhesion of keratinocytes in the basal and immediate suprabasal layers of stratified squamous epithelia. The pathology, at least partially, may depend on protease release from keratinocytes, but might also result from antibodies interfering with an adhesion function of Dsg3. However, a direct role of desmogleins in cell adhesion has not been shown. To test whether Dsg3 mediates adhesion, we genetically engineered mice with a targeted disruption of the DSG3 gene. DSG3 -/- mice had no DSG3 mRNA by RNase protection assay and no Dsg3 protein by immunofluorescence (IF) and immunoblots. These mice were normal at birth, but by 8-10 d weighed less than DSG3 +/- or +/+ littermates, and at around day 18 were grossly runted. We speculated that oral lesions (typical in PV patients) might be inhibiting food intake, causing this runting. Indeed, oropharyngeal biopsies showed erosions with histology typical of PV, including suprabasilar acantholysis and "tombstoning" of basal cells. EM showed separation of desmosomes. Traumatized skin also had crusting and suprabasilar acantholysis. Runted mice showed hair loss at weaning. The runting and hair loss phenotype of DSG3 -/- mice is identical to that of a previously reported mouse mutant, balding (bal). Breeding indicated that bal is coallelic with the targeted mutation. We also showed that bal mice lack Dsg3 by IF, have typical PV oral lesions, and have a DSG3 gene mutation. These results demonstrate the critical importance of Dsg3 for adhesion in deep stratified squamous epithelia and suggest that pemphigus autoantibodies might interfere directly with such a function.

The genetic variation in the tenomodulin gene is associated with serum total and LDL cholesterol in a body size-dependent manner.

We have reported that the sequence variation in the tenomodulin (TNMD) gene is associated with the risk of type 2 diabetes (T2DM), central obesity and serum levels of systemic immune mediators in the Finnish Diabetes Prevention Study (DPS), which is a longitudinal lifestyle intervention study on 522 middle-aged persons with impaired glucose tolerance (IGT). The aim of this study was to investigate whether the association with T2DM, observed in the DPS could be replicated in a larger, cross-sectional population-based random sample of 5298 men (3020 with normoglycaemia, 984 with impaired fasting glucose, 436 with IGT and 811 with T2DM) from the region of Kuopio, eastern Finland. To further explore the putative mechanisms linking TNMD to T2DM and metabolic syndrome, we studied the associations of TNMD sequence variation with lipid abnormalities characteristic to metabolic syndrome. The association with T2DM risk was not replicated, but significant associations were found with serum low-density lipoprotein and total cholesterol in a body mass index-dependent manner. These associations were also observed in the men of DPS, whereas in women these associations were not significant. These results from two independent study populations suggest that the genetic variation in TNMD could modulate cholesterol metabolism in obese men.

Weight reduction modulates expression of genes involved in extracellular matrix and cell death: the GENOBIN study.

OBJECTIVE: Lifestyle and genetic factors interact in the development of obesity and the metabolic syndrome. The molecular mechanisms underlying the beneficial dietary modifications are, however, unclear. We aimed to examine the effect of the long-term moderate weight reduction on gene expression in adipose tissue (AT) and to identify genes and gene clusters responsive to treatment and thereby likely contributing to the development of the metabolic syndrome. DESIGN: Randomized controlled and individualized weight reduction intervention. SUBJECTS: Forty-six subjects with impaired fasting glycemia or impaired glucose tolerance and features of metabolic syndrome, aged 60+/-7 years were randomized either to a weight reduction (WR) (n=28) or a control (n=18) group lasting for 33 weeks. MEASUREMENTS: Oral and intravenous glucose tolerance tests and subcutaneous AT biopsies were performed before and after the intervention. Gene expression of AT was studied using microarray technology in subgroups of WR (with weight reduction > or =5%, n=9) and control group (n=10). The results were confirmed using quantitative PCR. RESULTS: In the WR group, glucose metabolism improved. Moreover, an inverse correlation between the change in S (I) and the change in body weight was found (r=-0.44, P=0.026). Downregulation of gene expression (P<0.01) involving gene ontology groups of extracellular matrix and cell death was seen. Such changes did not occur in the control group. The tenomodulin-gene was one of the most downregulated genes (-39+/-16%, P<0.0001). Moreover, its expression correlated with insulin sensitivity (r=-0.34, P=0.005) before the intervention and with body adiposity both before (r=0.42, P=0.007) and after (r=0.30, P=0.056) the intervention. CONCLUSION: Genes regulating the extracellular matrix and cell death showed a strong downregulation after long-term weight reduction. This likely reflects a new stable state at the molecular level in AT. Further studies are warranted to elucidate the mechanisms of these genetic factors.

Epidermolytic hyperkeratosis (bullous congenital ichthyosiform erythroderma). Genetic linkage to chromosome 12q in the region of the type II keratin gene cluster.

Epidermolytic hyperkeratosis (EHK) is an autosomal dominant genodermatosis characterized by hyperkeratosis and blistering of the skin. Histopathology demonstrates suprabasilar blister formation with aggregation of tonofilaments. In this study, we tested the hypothesis that the EHK phenotype is linked to one of the suprabasilar keratins (KRT10 or KRT1) present in the types I and II keratin gene clusters in chromosomes 17q and 12q, respectively. For this purpose, Southern hybridizations were performed with DNA from a large kindred with EHK, consisting of 11 affected individuals in three generations. Segregation analysis with markers flanking the keratin gene clusters demonstrated linkage (Z = 3.61 at theta = 0) to a locus on 12q, while markers on 17q were excluded. These data implicate KRT1, the type II keratin expressed in suprabasilar keratinocytes, as a candidate gene in this family with EHK.

A homozygous nonsense mutation in the PLEC1 gene in patients with epidermolysis bullosa simplex with muscular dystrophy.

Plectin is a widely expressed cytomatrix component involved in the attachment of the cytoskeleton to the plasma membrane. We have recently reported that the skin and muscles of three patients affected by epidermolysis bullosa simplex with muscular dystrophy (MD-EBS), a genetic disorder characterized by skin blistering associated with muscle involvement, are not reactive with antibodies specific to plectin. We demonstrated that in the skin, lack of plectin leads to failure of keratin filaments to connect to the plasma membrane via the hemidesmosomes, whereas in the muscle the deficient expression of the molecule correlates with an aberrant localization of desmin in the muscle fibers. In this study we demonstrate that in a MD-EBS kindred with two affected members, the disease results from a homozygous nonsense mutation in the plectin (PLEC1) gene leading to a premature stop codon (CGA to TGA) and decay of the aberrant plectin messenger RNA. The segregation of the mutated allele implicates the mutation in the pathology of the disorder. These results confirm the critical role of plectin in providing cell resistance to mechanical stresses both in the skin and the muscle.

Chernobyl exposure as stressor during pregnancy and behaviour in adolescent offspring.

OBJECTIVE: Research in animals has shown that exposure to stressors during pregnancy is associated with offspring behavioural disorders. We aimed to study the effect of in utero exposure to the Chernobyl disaster in 1986, and maternal anxiety presumably associated with that exposure, on behaviour disorder observed at age 14. METHOD: Exposed (n = 232) and non-exposed Finnish twins (n = 572) were compared. A semi-structured interview was used to assess lifetime symptoms of depression, generalized anxiety disorder, attention deficit hyperactivity disorder, conduct disorder and oppositional defiant disorder symptoms. RESULTS: Adolescents who were exposed from the second trimester in pregnancy onwards, had a 2.32-fold risk (95% CI: 1.13-4.72) of having lifetime depression symptoms, an increased risk of fulfilling DSM-III-R criteria of a major depressive disorder (OR = 2.48, 95% CI: 1.06-5.7), and a 2.01-fold risk (95% CI: 1.14-3.52) of having attention deficit hyperactivity disorder symptoms. CONCLUSION: Perturbations in fetal brain development during the second trimester may be associated with the increased prevalence of depressive and attention deficit hyperactivity disorder symptoms.

Molecular genetics of pseudoxanthoma elasticum: a metabolic disorder at the environment-genome interface?

Pseudoxanthoma elasticum (PXE) is a relatively rare heritable disorder affecting the skin, eyes and cardiovascular system, with considerable morbidity and mortality. The disease affects the elastic fibers of affected organs, which become progressively calcified. Thus, PXE has been considered as a prototypic heritable connective tissue disorder affecting the elastic fiber system. Recently, PXE has been linked to mutations in the MRP6/ABCC6 gene, a member of the ABC transporter family, expressed primarily in the liver and the kidneys. This information, together with clinical observations suggesting environmental, hormonal and/or dietary modulation of the disease, raises the intriguing possibility that PXE is a primary metabolic disorder at the environment-genome interface.

The genodermatoses: candidate diseases for gene therapy.

Tremendous progress has been made in understanding the genetic basis of different forms of genodermatoses, a group of heritable diseases displaying a spectrum of phenotypic manifestations and clinical severity. The information about the underlying mutations in the candidate gene/protein systems has provided the basis for initial development of cutaneous gene therapy, and these heritable conditions appear to serve as appropriate candidate diseases for such efforts. Because of its accessibility and the fact that resident skin cells, such as epidermal keratinocytes and dermal fibroblasts, can be readily propagated in culture, skin serves as an appropriate target tissue for gene therapy. Various strategic considerations, including the use of in vivo or ex vivo approaches, gene replacement versus gene repair, utilization of different delivery systems, etc., require careful prioritization depending on the type of mutations and their pathogenetic consequences at the mRNA and protein levels.

Molecular basis of the dystrophic and junctional forms of epidermolysis bullosa: mutations in the type VII collagen and kalinin (laminin 5) genes.

Epidermolysis bullosa (EB) is a group of heritable mechano-bullous skin diseases classified into three major categories, the simplex, junctional, and dystrophic forms, on the basis of the level of tissue separation within the dermal-epidermal basement membrane zone. Approaches of molecular biology have demonstrated that these three different forms of EB result from mutations in distinct genes: the simplex forms are due to mutations in the genes encoding keratins 5 and 14 expressed in basal keratinocytes; the junctional forms are associated with mutations in the kalinin/laminin 5 genes; and the dystrophic forms result from mutations in the type VII collagen gene (COL7A1). In this overview, we summarize our recent discoveries of pathogenic mutations in COL7A1, including premature termination codons that result in the severe, mutilating (Hallopeau-Siemens) type of recessive dystrophic EB and a glycine substitution in the collagenous region resulting in dominant dystrophic EB. Furthermore, we present evidence that implicates mutations in the kalinin/laminin 5 gamma 2 chain gene (LAMC2) in some forms of junctional EB. This information has provided the basis for DNA-based prenatal diagnosis during the first trimester of gestation, and sets the stage for the application of gene therapy to these devastating skin diseases in the future.

Compound heterozygosity for nonsense and missense mutations in the LAMB3 gene in nonlethal junctional epidermolysis bullosa.

Mutations in the genes encoding laminin 5 (LAMA3, LAMB3, and LAMC2) have been delineated in the autosomal recessive blistering skin disorder, junctional epidermolysis bullosa, particularly in the lethal (Herlitz) variant. In this study, we searched for mutations in these genes in two patients with nonlethal forms of junctional epidermolysis bullosa using polymerase chain reaction amplification of genomic DNA, followed by heteroduplex analysis and direct automated nucleotide sequencing. Both patients were found to be compound heterozygotes for the same nonsense mutation on one LAMB3 allele, and different missense mutations on the other LAMB3 allele. The combination of nonsense and a missense mutation in the LAMB3 gene appears to be important in determining the milder clinical phenotype in some cases of the nonlethal forms of junctional epidermolysis bullosa involving abnormalities in laminin 5.

Novel COL7A1 mutations in dystrophic forms of epidermolysis bullosa.

Mutations in the type VII collagen gene (COL7A1) have been shown to underlie different variants of dystrophic epidermolysis bullosa (DEB). Examination of the genetic database indicates that most of the mutations are family specific, with few recurrent mutations. To facilitate further refinement of genotype/phenotype correlations in DEB, we have examined a cohort of nine families with DEB (seven recessively and two dominantly inherited) by a mutation detection strategy based on polymerase chain reaction amplification of COL7A1 genomic sequences, followed by heteroduplex scanning and direct nucleotide sequencing. The results revealed 16 allelic mutations, 11 of them being novel, previously unpublished. The genetic information was also used for prenatal testing in a family at risk for recurrence of a severe, Hallopeau-Siemens type of RDEB. These data contribute to the expanding database of COL7A1 mutations in DEB.