Amnionless, essential for mouse gastrulation, is mutated in recessive hereditary megaloblastic anemia.

The amnionless gene, Amn, on mouse chromosome 12 encodes a type I transmembrane protein that is expressed in the extraembryonic visceral layer during gastrulation. Mice homozygous with respect to the amn mutation generated by a transgene insertion have no amnion. The embryos are severely compromised, surviving to the tenth day of gestation but seem to lack the mesodermal layers that normally produce the trunk. The Amn protein has one transmembrane domain separating a larger, N-terminal extracellular region and a smaller, C-terminal cytoplasmic region. The extracellular region harbors a cysteine-rich domain resembling those occurring in Chordin, found in Xenopus laevis embryos, and Sog, found in Drosophila melanogaster. As these cysteine-rich domains bind bone morphogenetic proteins (Bmps), it has been speculated that the cysteine-rich domain in Amn also binds Bmps. We show that homozygous mutations affecting exons 1-4 of human AMN lead to selective malabsorption of vitamin B12 (a phenotype associated with megaloblastic anemia 1, MGA1; OMIM 261100; refs. 5,6) in otherwise normal individuals, suggesting that the 5' end of AMN is dispensable for embryonic development but necessary for absorption of vitamin B12. When the 5' end of AMN is truncated by mutations, translation is initiated from alternative downstream start codons.

Cerebroretinal microangiopathy with calcifications and cysts: characterization of the skeletal phenotype.

Cerebral cysts and calcifications with leukoencephalopathy and retinal vascular abnormalities are diagnostic hallmarks of cerebroretinal microangiopathy with calcifications and cysts (CRMCC). Previous studies have suggested that skeletal involvement is also common, but its characteristics remain unknown. This study aimed to assess the skeletal phenotype in CRMCC. All Finnish patients with features consistent with CRMCC and for whom radiographs were available were included. Clinical information pertinent to the skeletal phenotype was collected from hospital records, and all plain radiographs were reviewed for skeletal features. Bone mineral density (BMD) was measured by DXA. In one patient, bone biopsies were obtained for bone histology and histomorphometric analyses. The LRP5 gene was analyzed for mutations by direct sequencing. Our results show that the skeletal phenotype in CRMCC includes (1) compromised longitudinal growth pre- and postnatally, (2) generalized osteopenia or early onset low turnover osteoporosis with fragility fractures, and (3) metaphyseal abnormalities that may lead to limb deformities such as short femoral neck or genua valga. DXA measurements in three patients showed low BMD, and bone biopsies in the fourth patient with pathological fractures and impaired fracture healing showed low-turnover osteoporosis, with reduced osteoclast and osteoblast activity. Direct sequencing of all LRP5 coding exons and exon-intron boundaries in six patients with CRMCC revealed no putative mutations. We conclude that the CRMCC-associated bone disease is characterized by low BMD and pathological fractures with delayed healing, metaphyseal changes, and short stature pre- and postnatally. LRP5 is not a disease-causing gene in CRMCC.

Low density lipoprotein receptor-related protein 5 (LRP5) mutations and osteoporosis, impaired glucose metabolism and hypercholesterolaemia.

OBJECTIVE: Mutations in the low-density lipoprotein receptor-related protein 5 gene (LRP5) underlie osteoporosis-pseudoglioma syndrome. Animal models implicate a role for LRP5 in lipid and glucose homeostasis. The objective was to evaluate metabolic consequences of LRP5 mutations in humans. DESIGN AND PATIENTS: Thirteen Finnish individuals with homozygous or heterozygous LRP5 mutations were assessed for bone health, glucose and lipid metabolism, and for serum serotonin concentration. Results were compared with findings in family members without mutations. MEASUREMENTS: Bone mineral density (BMD), vertebral morphology, oral and intravenous glucose tolerance tests, lipid profile and serum serotonin concentrations. RESULTS: Two individuals were homozygous for R570W, one compound heterozygous for R570W and R1036Q, and 10 were heterozygous (six for R570W, three for R1036Q and one for R925C). Subjects with two LRP5 mutations had multiple spinal fractures and low BMD. Subjects with one mutation had significantly lower median lumbar spine (P = 0.004) and femoral neck (P = 0.005) BMD Z-scores, and more often vertebral fractures than the 18 individuals without mutations. Of the 12 subjects with LRP5 mutation six had diabetes and one had impaired glucose tolerance. Intravenous glucose tolerance tests suggested impaired beta-cell function; no insulin resistance was observed. Prevalence of hypercholesterolaemia was similar in mutation positive and negative subjects. Serum serotonin concentrations showed a trend towards higher concentrations in subjects with LRP5 mutation. CONCLUSIONS: We found high prevalence of osteoporosis and abnormal glucose metabolism in subjects with LRP5 mutation(s). Further studies are needed to establish the role of LRP5 in glucose and lipid metabolism.

Mechanisms behind bottom-up effects: eutrophication increases fecundity by shortening the interspawning interval in stickleback.

Anthropogenic eutrophication is altering aquatic environments by promoting primary production. This influences the population dynamics of consumers through bottom-up effects, but the underlying mechanisms and pathways are not always clear. To evaluate and mitigate effects of eutrophication on ecological communities, more research is needed on the underlying factors. Here we show that anthropogenic eutrophication increases population fecundity in the threespine stickleback (Gasterosteus aculeatus) by increasing the number of times females reproduce-lifetime fecundity-rather than instantaneous fecundity. When we exposed females to nutrient-enriched waters with enhanced algal growth, their interspawning interval shortened but the size of their egg clutches, or the size of their eggs, did not change. The shortening of the interspawning interval was probably caused by higher food intake, as algae growth promotes the growth of preferred prey populations. Enhanced female lifetime fecundity could increase offspring production and, hence, influence population dynamics. In support of this, earlier studies show that more offspring are emerging in habitats with denser algae growth. Thus, our results stress the importance of considering lifetime fecundity, in addition to instantaneous fecundity, when investigating the impact of human-induced eutrophication on population processes. At a broader level, our results highlight the importance of following individuals over longer time spans when evaluating the pathways and processes through which environmental changes influence individual fitness and population processes.

LRP5 in premature adrenarche and in metabolic characteristics of prepubertal children.

OBJECTIVE: Premature adrenarche (PA) is associated with unfavourable metabolic characteristics. We hypothesized that genetic variation in low density lipoprotein (LDL) receptor-related protein 5 (LRP5), which is involved in Wnt signalling in the adrenal cortex and in cholesterol metabolism, plays a role in the pathogenesis of PA. DESIGN AND PATIENTS: We performed a cross-sectional association study in 73 Finnish children with PA and 97 age- and gender-matched healthy controls. MEASUREMENTS: LRP5 genotypes were determined by direct sequencing. Single-marker associations with clinical-metabolic characteristics, including adrenocortical function, glucose tolerance and lipid profile, were examined with age and gender as covariates. RESULTS: Nineteen single nucleotide polymorphisms (SNPs) in LRP5 were found in the 170 children. No significant differences in the genotype distributions were observed between the PA and control groups. SNPs A1330V and N740N were associated with higher serum dehydroepiandrosterone sulphate (DHEAS) levels in the control subjects (A/A vs. A/a; mean 0.8 vs. 1.4 micromol/l, P = 0.01). They were also associated with higher plasma levels of total (4.2 vs. 4.7 mmol/l, P = 0.02) and LDL cholesterol (2.4 vs. 2.9 mmol/l, P = 0.02) in the control group, as was SNP V1119V (P = 0.04 and P = 0.03, respectively). SNPs F549F and V1119V were associated with higher systolic blood pressure (P = 0.04 and P = 0.02, respectively). There were no differences in the parameters of glucose metabolism between the genotype groups. CONCLUSIONS: Genetic variation in LRP5 did not predispose to PA but was associated with metabolic characteristics, especially lipid profile, in healthy prepubertal children.

The A1330V polymorphism of the low-density lipoprotein receptor-related protein 5 gene (LRP5) associates with low peak bone mass in young healthy men.

INTRODUCTION: Polymorphisms in the gene coding for low-density lipoprotein receptor-related protein 5 (LRP5) contribute to variation in bone mass in the general population. Whether this is due to influence on bone mass acquisition or on bone loss thereafter has not been established. METHODS: We studied the association of LRP5 polymorphisms with peak bone mass in young men. The study included 235 Finnish men, aged 18.3 to 20.6 years. Lifestyle factors and fracture history were recorded. Bone mineral content (BMC), density (BMD) and scan area were measured for the lumbar spine and proximal femur by dual energy X-ray absorptiometry (DXA). Blood and urine were collected for determination of bone turnover markers, serum 25-OHD and PTH. Genomic DNA was extracted from peripheral blood for genetic analysis of LRP5. Ten single nucleotide polymorphisms in LRP5 were analyzed and correlated with bone parameters. RESULTS: Only the A1330V polymorphism of LRP5 significantly associated with bone parameters. In comparison with subjects with the AlaAla genotype (n=215), those with AlaVal genotype (n=20) had lower femoral neck BMC (P=0.029) and BMD (P=0.012), trochanter BMC (P=0.0067) and BMD (P=0.015), and total hip BMC (P=0.0044) and BMD (P=0.0089). Fracture history was similar for the genotypes. CONCLUSION: The polymorphic valine variant at position 1330 of LRP5 was significantly associated with reduced BMC and BMD values in healthy young Finnish men. The results provide evidence for the crucial role of LRP5 in peak bone mass acquisition.

Low-density lipoprotein receptor-related protein 5 (LRP5) variation in fracture prone children.

OBJECTIVE: Recent studies have confirmed that the low-density lipoprotein receptor-related protein 5 gene (LRP5), plays a role in bone mass accrual and in susceptibility to osteoporotic fractures in adults. This study evaluated whether LRP5 variation is implicated in childhood fractures. PATIENTS AND METHODS: During an epidemiological study on childhood fractures, comprising 1390 consecutive Finnish children with an acute fracture, we recruited fracture-prone 4-16 years old children, who had a history of at least two low-energy long bone fractures before age 10 years or three low-energy long bone fractures before age 16 years, and/or at least one low-energy vertebral compression fracture. A total of 72 (5.2%) children fulfilled these inclusion criteria; DNA samples were obtained for 66 of them. All 23 exons and exon-intron boundaries of the LRP5 gene were sequenced; the identified variants were analyzed in 235 healthy Finnish control samples. RESULTS: Sequencing revealed 15 coding region missense or silent variants with unknown functional consequences. No obvious loss-of-function mutations such as deletions, insertions, or changes resulting in premature termination codon or altered splicing were identified. Phenotyping of the proband and parents, and genotyping of the parents, in 9 families with novel or rare variants showed no obvious correlation between any of the LRP5 variants and fractures. CONCLUSIONS: Our study shows that in children LRP5 mutations are not a common cause of increased fractures. The observed rare LRP5 variants may together with unfavorable environmental and other genetic factors contribute to childhood fractures, but further studies are needed to confirm their functional significance and biological pathways through which this may occur. Our findings suggest that systematic LRP5 screening is not indicated in children with recurrent fractures.

Body composition and bone mineral density in children with premature adrenarche and the association of LRP5 gene polymorphisms with bone mineral density.

CONTEXT: Precocious increase in adrenal androgen production is the hallmark of premature adrenarche (PA). Adrenal androgens have anabolic properties. OBJECTIVE: The objective of the study was to test whether body composition and bone mineral density (BMD) are altered in PA and study whether genetic variation in low-density lipoprotein receptor-related protein 5 (LRP5) affects BMD in PA. DESIGN: This was a cross-sectional study. SETTING: The study was conducted at a university hospital. SUBJECTS AND MEASURES: The study included 126 prepubertal children (64 with PA, 10 boys; 62 non-PA controls, 10 boys). Femoral neck and lumbar spine areal and calculated volumetric BMD (dual energy X-ray absorptiometry), body composition (bioimpedance), serum 25-hydroxyvitamin D, and markers of bone turnover and calcium homeostasis were compared between the PA and control groups. Single-nucleotide polymorphisms of LRP5 were determined and associated with BMD. RESULTS: Children with PA had higher femoral neck and lumbar spine BMD(areal) than the controls (Z-score 0.56 vs. -0.09, P < 0.001, and 0.20 vs. -0.31, P = 0.009, respectively). However, the mean BMDs did not differ significantly between the groups when adjusted for height or bone size. BMD(areal) correlated strongly with height sd score in both groups. Among the PA children, LRP5 single-nucleotide polymorphism E644E minor variant was associated with lower and F549F minor variant with higher BMD. Total body fat mass, fat percent, serum PTH, and alkaline phosphatase concentrations were higher and 25-hydroxyvitamin D lower in the PA group. CONCLUSIONS: Prepubertal children with PA had higher BMD(areal) compared with healthy controls. This was mainly explained by their increased height. LRP5 polymorphisms may contribute to bone mass accrual in prepubertal PA children.

Cohen syndrome is caused by mutations in a novel gene, COH1, encoding a transmembrane protein with a presumed role in vesicle-mediated sorting and intracellular protein transport.

Cohen syndrome is an uncommon autosomal recessive disorder whose diagnosis is based on the clinical picture of nonprogressive psychomotor retardation and microcephaly, characteristic facial features, retinal dystrophy, and intermittent neutropenia. We have refined the critical region on chromosome 8q22 by haplotype analysis, and we report the characterization of a novel gene, COH1, that is mutated in patients with Cohen syndrome. The longest transcript (14,093 bp) is widely expressed and is transcribed from 62 exons that span a genomic region of approximately 864 kb. COH1 encodes a putative transmembrane protein of 4,022 amino acids, with a complex domain structure. Homology to the Saccharomyces cerevisiae VPS13 protein suggests a role for COH1 in vesicle-mediated sorting and transport of proteins within the cell.

Delineation of Cohen syndrome following a large-scale genotype-phenotype screen.

Cohen syndrome is an autosomal recessive condition associated with developmental delay, facial dysmorphism, pigmentary retinopathy, and neutropenia. The pleiotropic phenotype, combined with insufficient clinical data, often leads to an erroneous diagnosis and has led to confusion in the literature. Here, we report the results of a comprehensive genotype-phenotype study on the largest cohort of patients with Cohen syndrome assembled to date. We found 22 different COH1 mutations, of which 19 are novel, in probands identified by our diagnostic criteria. In addition, we identified another three novel mutations in patients with incomplete clinical data. By contrast, no COH1 mutations were found in patients with a provisional diagnosis of Cohen syndrome who did not fulfill the diagnostic criteria ("Cohen-like" syndrome). This study provides a molecular confirmation of the clinical phenotype associated with Cohen syndrome and provides a basis for laboratory screening that will be valuable in its diagnosis.