Amnionless Research Articles

A frameshift mutation in the cubilin gene (CUBN) in Beagles with Imerslund-Gräsbeck syndrome (selective cobalamin malabsorption)

Mammals are unable to synthesize cobalamin or vitamin B12 and rely on the uptake of dietary cobalamin. The cubam receptor expressed on the intestinal endothelium is required for the uptake of cobalamin from the gut. Cubam is composed of two protein subunits, amnionless and cubilin, which are encoded by the AMN and CUBN genes respectively. Loss-of-function mutations in either the AMN or the CUBN gene lead to hereditary selective cobalamin malabsorption or Imerslund-Gräsbeck syndrome (IGS). We investigated Beagles with IGS and resequenced the whole genome of one affected Beagle at 15× coverage. The analysis of the AMN and CUBN candidate genes revealed a homozygous deletion of a single cytosine in exon 8 of the CUBN gene (c.786delC). This deletion leads to a frameshift and early premature stop codon (p.Asp262Glufs*47) and is, thus, predicted to represent a complete loss-of-function allele. We tested three IGS-affected and 89 control Beagles and found perfect association between the IGS phenotype and the CUBN:c.786delC variant. Given the known role of cubilin in cobalamin transport, which has been firmly established in humans and dogs, our data strongly suggest that the CUBN:c.786delC variant is causing IGS in the investigated Beagles.

Detailed investigations of proximal tubular function in Imerslund-Gräsbeck syndrome

BackgroundImerslund-Gräsbeck Syndrome (IGS) is a rare genetic disorder characterised by juvenile megaloblastic anaemia. IGS is caused by mutations in either of the genes encoding the intestinal intrinsic factor-vitamin B12 receptor complex, cubam. The cubam receptor proteins cubilin and amnionless are both expressed in the small intestine as well as the proximal tubules of the kidney and exhibit an interdependent relationship for post-translational processing and trafficking. In the proximal tubules cubilin is involved in the reabsorption of several filtered plasma proteins including vitamin carriers and lipoproteins. Consistent with this, low-molecular-weight proteinuria has been observed in most patients with IGS. The aim of this study was to characterise novel disease-causing mutations and correlate novel and previously reported mutations with the presence of low-molecular-weight proteinuria.MethodsGenetic screening was performed by direct sequencing of the CUBN and AMN genes and novel identified mutations were characterised by in silico and/or in vitro investigations. Urinary protein excretion was analysed by immunoblotting and high-resolution gel electrophoresis of collected urines from patients and healthy controls to determine renal phenotype.ResultsGenetic characterisation of nine IGS patients identified two novel AMN frameshift mutations alongside a frequently reported AMN splice site mutation and two CUBN missense mutations; one novel and one previously reported in Finnish patients. The novel AMN mutations were predicted to result in functionally null AMN alleles with no cell-surface expression of cubilin. Also, the novel CUBN missense mutation was predicted to affect structural integrity of the IF-B12 binding site of cubilin and hereby most likely cubilin cell-surface expression. Analysis of urinary protein excretion in the patients and 20 healthy controls revealed increased urinary excretion of cubilin ligands including apolipoprotein A-I, transferrin, vitamin D-binding protein, and albumin. This was, however, only observed in patients where plasma membrane expression of cubilin was predicted to be perturbed.ConclusionsIn the present study, mutational characterisation of nine IGS patients coupled with analyses of urinary protein excretion provide additional evidence for a correlation between mutation type and presence of the characteristic low-molecular-weight proteinuria.

Genomic and Functional Analyses of the 2-Aminophenol Catabolic Pathway and Partial Conversion of Its Substrate into Picolinic Acid in Burkholderia xenovorans LB400

2-aminophenol (2-AP) is a toxic nitrogen-containing aromatic pollutant. Burkholderia xenovorans LB400 possess an amn gene cluster that encodes the 2-AP catabolic pathway. In this report, the functionality of the 2-aminophenol pathway of B. xenovorans strain LB400 was analyzed. The amnRJBACDFEHG cluster located at chromosome 1 encodes the enzymes for the degradation of 2-aminophenol. The absence of habA and habB genes in LB400 genome correlates with its no growth on nitrobenzene. RT-PCR analyses in strain LB400 showed the co-expression of amnJB, amnBAC, amnACD, amnDFE and amnEHG genes, suggesting that the amn cluster is an operon. RT-qPCR showed that the amnB gene expression was highly induced by 2-AP, whereas a basal constitutive expression was observed in glucose, indicating that these amn genes are regulated. We propose that the predicted MarR-type transcriptional regulator encoded by the amnR gene acts as repressor of the amn gene cluster using a MarR-type regulatory binding sequence. This report showed that LB400 resting cells degrade completely 2-AP. The amn gene cluster from strain LB400 is highly identical to the amn gene cluster from P. knackmussi strain B13, which could not grow on 2-AP. However, we demonstrate that B. xenovorans LB400 is able to grow using 2-AP as sole nitrogen source and glucose as sole carbon source. An amnBA − mutant of strain LB400 was unable to grow with 2-AP as nitrogen source and glucose as carbon source and to degrade 2-AP. This study showed that during LB400 growth on 2-AP this substrate was partially converted into picolinic acid (PA), a well-known antibiotic. The addition of PA at lag or mid-exponential phase inhibited LB400 growth. The MIC of PA for strain LB400 is 2 mM. Overall, these results demonstrate that B. xenovorans strain LB400 posses a functional 2-AP catabolic central pathway, which could lead to the production of picolinic acid.

Imerslund-Gräsbeck syndrome in a 25-month-old Italian girl caused by a homozygous mutation in AMN

Imerslund-Gräsbeck syndrome is a rare autosomal recessive disorder, characterized by vitamin B12 deficiency due to selective malabsorption of the vitamin and usually results in megaloblastic anemia appearing in childhood. It is responsive to parenteral vitamin B12 therapy.The estimated prevalence (calculated based on Scandinavian data) is less than 6:1,000,000. However, many cases may be misdiagnosed.When there is reasonable evidence to suspect that a patient suffers from IGS, a new and straightforward approach to diagnosis is mutational analysis of the appropriate genes. We report for the first time the case of a girl of Italian ancestry with IGS genetically confirmed by the detection of a homozygous missense mutation in the AMN gene (c.208-2 A > G).

An exon 53 frameshift mutation in CUBN abrogates cubam function and causes Imerslund-Gräsbeck syndrome in dogs

Cobalamin malabsorption accompanied by selective proteinuria is an autosomal recessive disorder known as Imerslund-Gräsbeck syndrome in humans and was previously described in dogs due to amnionless (AMN) mutations. The resultant vitamin B12 deficiency causes dyshematopoiesis, lethargy, failure to thrive, and life-threatening metabolic disruption in the juvenile period. We studied 3 kindreds of border collies with cobalamin malabsorption and mapped the disease locus in affected dogs to a 2.9Mb region of homozygosity on canine chromosome 2. The region included CUBN, the locus encoding cubilin, a peripheral membrane protein that in concert with AMN forms the functional intrinsic factor-cobalamin receptor expressed in ileum and a multi-ligand receptor in renal proximal tubules. Cobalamin malabsorption and proteinuria comprising CUBN ligands were demonstrated by radiolabeled cobalamin uptake studies and SDS-PAGE, respectively. CUBN mRNA and protein expression were reduced ~10 fold and ~20 fold, respectively, in both ileum and kidney of affected dogs. DNA sequencing demonstrated a single base deletion in exon 53 predicting a translational frameshift and early termination codon likely triggering nonsense mediated mRNA decay. The mutant allele segregated with the disease in the border collie kindred. The border collie disorder indicates that a CUBN mutation far C-terminal from the intrinsic factor-cobalamin binding site can abrogate receptor expression and cause Imerslund-Gräsbeck syndrome.

From bowel to kidneys: the role of cubilin in physiology and disease

Cubilin is a large endocytic receptor serving such diverse functions as the intestinal absorption of the intrinsic factor-B(12) complex and the renal proximal tubule reabsorption of filtered proteins including albumin, transferrin, vitamin D-binding protein and other important plasma carriers. Cubilin is a structurally unique, peripheral membrane protein, which depends on the membrane protein amnionless (AMN) for correct apical translocation. In addition, AMN appears important for efficient internalization of intrinsic factor-B(12) in the intestine, whereas in the proximal tubule cubilin interacts with another endocytic receptor, megalin, for effective reabsorption. The importance of cubilin has been demonstrated in several animal models of cubilin deficiency as well as in a variety of human diseases. Recent demonstration of cubilin in podocytes from various species awaits further clarification with respect to the functional role as well as its role in pathology.

Cubilin and Amnionless Mediate Protein Reabsorption in Drosophila Nephrocytes

The insect nephrocyte and the mammalian glomerular podocyte are similar with regard to filtration, but it remains unclear whether there is an organ or cell type in flies that reabsorbs proteins. Here, we show that the Drosophila nephrocyte has molecular, structural, and functional similarities to the renal proximal tubule cell. We screened for genes required for nephrocyte function and identified two Drosophila genes encoding orthologs of mammalian cubilin and amnionless (AMN), two major receptors for protein reabsorption in the proximal tubule. In Drosophila, expression of dCubilin and dAMN is specific to nephrocytes, where they function as co-receptors for protein uptake. Targeted expression of human AMN in Drosophila nephrocytes was sufficient to rescue defective protein uptake induced by dAMN knockdown, suggesting evolutionary conservation of Cubilin/AMN co-receptors function from flies to humans. Furthermore, we found that Cubilin/AMN-mediated protein reabsorption is required for the maintenance of nephrocyte ultrastructure and fly survival under conditions of toxic stress. In conclusion, the insect nephrocyte combines filtration with protein reabsorption, using evolutionarily conserved genes and subcellular structures, suggesting that it can serve as a simplified model for both podocytes and the renal proximal tubule.

How can cobalamin injections be spaced in long-term therapy for inborn errors of vitamin B12 absorption?

Inborn errors of cobalamin (Cbl, vitamin B12) absorption include hereditary intrinsic factor deficiency (HIFD) and Imerslund–Gräsbeck disease (IGD). HIFD is secondary to mutations in the HIF gene while IGD is due to mutations in one of the 2 subunits of the intrinsic factor receptor that is cubilin (CUBN) or amnionless (AMN). These disorders lead to intracellular Cbl depletion which in turn causes megaloblastic bone marrow failure, accumulation of homocysteine and methylmalonic acid (MMA), and methionine depletion. The clinical presentation reflects Cbl deficiency, with gastrointestinal symptoms, pancytopenia, and megaloblastic anemia. Mixed proteinuria, when it is present is strongly suggestive of IGD. Accurate diagnosis is always an emergency because early detection and treatment with life-long parenteral pharmacological doses of hydroxocobalamin are life saving and prevent further deterioration. However, the optimal frequency for cobalamin injections as a maintenance therapy is poorly reported. In order to evaluate the optimal maintenance schedule of cobalamin injections, we retrospectively collected clinical, biological, molecular and treatment data on 7 patients affected with congenital Cbl malabsorption. Unlike previous recommendations, we showed that a maintenance dosage of 1mg cobalamin twice a year was enough to ensure a normal clinical status and keep the hematological and metabolic parameters in the normal range. These data suggest that patients affected with inborn errors of cobalamin absorption may be safely long-term treated with cobalamin injections every 6months with careful follow-up of hematological and metabolic parameters. This maintenance regime is beneficial because the patients’ quality of life improves.

Expression of amnionless in mouse testes and Leydig cells

Vitamin B(12) (cobalamin) deficiency results in atrophy of seminiferous tubules and aplasia of spermatozoa and spermatid. The transmembrane protein amnionless (AMN) directs endocytosis of cubilin with its ligand, contributing to intrinsic factor-vitamin B(12) absorption. To understand vitamin B(12) transport in testis, we analysed AMN expression in developing mouse testes and in Leydig cells and speculated the possible role of AMN in testis. In testes, Amn mRNA levels were low until 14 days post partum (pp) and markedly increased from puberty onwards. In the interstitium, Amn mRNA levels were low at 14 days pp and increased at puberty (28 days pp) together with 3-beta-hydroxysteroid dehydrogenase type 6 mRNA. Strong AMN immunoreactivity was observed in early spermatocytes from 7 days pp, suggesting that AMN participates in meiosis. In Leydig cells, AMN was not observed until 14 days pp but was strongly expressed after 28 days pp, suggesting a positive relationship between AMN expression and functional differentiation of adult Leydig cells. Together, AMN may participate in meiosis in early spermatocytes and in functional differentiation of adult Leydig cells through the mediation of vitamin B(12) transport in the mouse testes. This is the first report on AMN expression in the germ cells and soma of mammalian testes.

Luminal expression of cubilin is impaired in Imerslund-Grasbeck syndrome with compound AMN mutations in intron 3 and exon 7

Juvenile megaloblastic anaemia 1 (OMIM # 261100) is a rare autosomic disorder characterized by selective cobalamin mal-absorption and inconstant proteinuria produced by mutations in either CUBN or AMN genes. Amnionless, the gene product of AMN, is a transmembrane protein that binds tightly to the N-terminal end of cubilin, the gene product of CUBN. Cubilin binds to intrinsic factor-cobalamin complex and is expressed in the distal intestine and the proximal renal tubule. We report a compound AMN heterozygosity with c.742C>T, p.Gln248X and c.208-2A>G mutations in 2 siblings that led to premature termination codon in exon 7 and exon 6, respectively. It produced a dramatic decrease in receptor activity in urine, despite absence of CUBN mutation and normal affinity of the receptor for intrinsic factor binding. Heterozygous carriers for c.742T and c.208-2G had no pathological signs. These results indicate that amnionless is essential for the correct luminal expression of cubilin in humans.

Amnionless Expression in the Mouse Testis and Leydig Cells and Its Change in Leydig Cell Hyperplasia.

In testis, vitamin B12 (cobalmin)-deficiency resulted in atrophy of seminiferous tubules and aplasia of sperms and spermatids. Deficiency in vitamin B12 decreased in methionine synthase activity. Vitamin B12 involves in DNA synthesis as it participates in methionine synthase followed by thymidine synthesis. Amnionless (Amn), a transmembrane protein directs endocytosis of cubilin with its ligand, contributing intrinsic factor-vitamin B12 absorption. Expressional profile from data base revealed that Amn is considerably expressed in kidney, small intestine, placenta, and testis in mouse. In an effort to understand vitamin B12 transport to the testicular cells, we analyzed the Amn gene expression in the developing mouse testis and Leydig cells. In the meantime, change of Amn expression in Leydig cell hyperplasia by flutamide an antiandrogen was examined in testis. In mouse testis, Amn mRNA levels were low until 14 days post partum (pp), markedly increased at puberty onwards. In the interstitium, Amn mRNA levels were low until 14 days pp, markedly increased together with 3β-hydroxysteroid dehydrogenase type VI mRNA at puberty (28 days pp), and further increased at 56 days pp. AMN immunoreactivity was found weakly in Sertoli cells throughout the testis development. Early spermatocytes showed strong AMN immunoreactivity at 14 days pp onwards, suggesting that AMN participates in the early meiosis. In Leydig cells, AMN immunoreactivity was not found until 14 days pp but strongly expressed at 28 days pp onwards. Quantitative image analysis revealed that AMN immunoreactivity in Leydig cells was increased by 2.8 fold between 28 and 56 days pp. Mean size of AMN-positive Leydig cells was increased by 1.8 fold between 14 and 28 days pp and 1.5 fold between 28 and 56 days pp. This suggests positive relationship between AMN expression and functional differentiation of adult Leydig cells. In adult male mice, oral administration of flutamide (100 mg/kg/day) for 10 days induced a Leydig cell hyperplasia in mouse testis in which Amn mRNA and AMN immunoreactivity were increased in Leydig cells, suggesting that AMN may participate in the pathogenesis of Leydig cell hyperplasia. In adult Leydig cells primary culture, flutamide (100 nM) increased Amn mRNA expression, suggesting that Amn gene expression is negatively coupled with androgen receptor activation. In conclusion, AMN may participate in the meiosis of early spermatocytes and functional differentiation of adult Leydig cells via mediating the vitamin B12 transport in mouse testis. In addition, AMN may participate in the Leydig cell hyperplasia via promoting cell proliferation in Leydig cell via stimulating DNA synthesis. This is the first report on the AMN expression in male germ cells and somatic cells in mammalian testis. (poster)

Molecular Study of Proteinuria in Patients Treated with B12 Supplements: Do Not Forget Megaloblastic Anemia Type 1

Background/Aims: Current consensus supports the notion that proteinuria is a marker of renal disease with prognostic implications. Whereas most chronic kidney disease patients with proteinuria would often require antiproteinuric agents, there are some exceptions. Megaloblastic anemia type 1 (MGA1) is characterized by megaloblastic anemia due to congenital selective vitamin B₁₂ malabsorption and proteinuria. In the present study, we describe 2 Israeli Jewish patients with MGA1 and isolated proteinuria. Methods: Because of their origin, the patients were screened for the presence of the already studied Tunisian AMN mutation, by direct sequencing the corresponding region from genomic DNA. PCR products were purified and sequenced. Results: Genomic DNA sequencing of the AMN gene of both patients confirmed that the acceptor splice site in intron 3 was changed from CAG to CGG (208-2A→G). Conclusion: We determined the molecular basis of MGA1 in both patients and discuss the involvement of the cubilin/AMN complex in this pathology and its role in the development of the proteinuria. We also discuss the questionable significance of antiproteinuric treatment for these patients.

Vitamin B12 deficiency in a 15-year old boy due to mutations in the intrinsic factor gene, GIF

The discoveries of new genes involved in cobalamin assimilation and metabolism has increased our understanding of the rare inborn errors of cobalamin metabolism (Quadros 2009). The following case documents a compound heterozygote mutation in the gastric intrinsic factor (GIF) gene, with a previously described mutation (c.79+1G>A) and a novel mutation (c.290T>C; M97T) leading to a megaloblastic anaemia in an adolescent.

Exploiting cell‐free systems: Implementation and debugging of a system of biotransformations

The orchestration of a multitude of enzyme catalysts allows cells to carry out complex and thermodynamically unfavorable chemical conversions. In an effort to recruit these advantages for in vitro biotransformations, we have assembled a 10-step catalytic system-a system of biotransformations (SBT)-for the synthesis of unnatural monosaccharides based on the versatile building block dihydroxyacetone phosphate (DHAP). To facilitate the assembly of such a network, we have insulated a production pathway from Escherichia coli's central carbon metabolism. This pathway consists of the endogenous glycolysis without triose-phosphate isomerase to enable accumulation of DHAP and was completed with lactate dehydrogenase to regenerate NAD(+). It could be readily extended for the synthesis of unnatural sugar molecules, such as the unnatural monosaccharide phosphate 5,6,7-trideoxy-D-threo-heptulose-1-phosphate from DHAP and butanal. Insulation required in particular inactivation of the amn gene encoding the AMP nucleosidase, which otherwise led to glucose-independent DHAP production from adenosine phosphates. The work demonstrates that a sufficiently insulated in vitro multi-step enzymatic system can be readily assembled from central carbon metabolism pathways.

Identification of Amnionless Gene as an Estrogen/ER Alpha-Regulated Gene in the Mouse Pituitary.

Estrogen plays a key role in regulating gonadotropin secretion from the pituitary through both negative and positive feedback pathways. However, the direct mechanisms of this regulation have yet to be defined. We hypothesize that the positive feedback effect of estrogen is achieved by ERα-mediated transcriptional regulation of cellular components needed for LH vesicle transport and secretion of its components into the bloodstream, thus equipping cells with the secretory machinery required for the LH surge. To identify these ERα-regulated components of LH secretion, total RNA was extracted from the pituitaries of cycling mice in metestrus or proestrus (low and high levels of circulating estrogen, respectively), ovariectomized (OVX) WT mice treated with vehicle or estrogen, and OVX ERαKO mice treated with vehicle or estrogen, and subjected to microarray analysis. Overall, 183 genes were found to be differentially expressed in an estrogen/ERα-dependent manner. In this study, we characterized the expression pattern of a gene (amn) that codes for Amnionless (Amn), a type I transmembrane protein known to be involved in vitamin B12 absorption by distal intestinal epithelia and proximal tubules of the kidney. The amn gene was selected as one gene of interest due to significant effect of estrogen treatment on transcription levels and the novelty of its characterization in the pituitary. The microarray analysis revealed a 30-fold increase of amn transcription in proestrus compared to metestrus, and a fold change of 25 in OVX WT mice with estrogen compared to vehicle treatment. ERαKO mice showed no increase in transcription of amn with estrogen treatment. This pattern of amn expression was confirmed using RT-PCR, showing a 9 fold increase in transcription levels in proestrus versus metestrus, and 16-fold increase in the OVX WT estrogen treated compared to vehicle treated groups. Immunohistochemistry confirmed that protein levels increased concurrently with mRNA levels in proestrus versus metestrus, but not in ERαKO mice. In silico analysis of the amn proximal promoter reveals 8 putative EREs within 2 kilo bases upstream of the start codon, evidence of a possible direct regulation of amn expression by the classical ERα pathway, which will be determined by ChIP assay. Currently, we are determining the functional significance of ERα-regulated amn expression in LH secretion using in vitro models. Both primary pituitary cells and isolated gonadotroph cells are treated with anti-Amnionless antibody, in order to block the functional capacity of the protein, followed by GnRH treatment. This experiment in the primary pituitary cells will allow us to determine if Amn in the pituitary plays a critical role in facilitating LH secretion, and the experiment in isolated gonadotroph cells will clarify if the effect on LH secretion is due to Amn in gonadotrophs themselves, or secondarily due to Amn expression in other cell types of the pituitary. For isolation of gonadotroph cells, we have generated ROSA-GFP CgaCre mice, who express Cre, and thus GFP, specifically in gonadotroph. Using flow cytometry, live gonadotrophs can be segregated from other cell types of the pituitary. Future directions will be aimed at elucidation of the role of Amn in preparing the gonadotroph for LH secretion in relation to the well-known function of Amn in facilitating vitamin B12 absorption. In summary, the gene encoding the Amnionless protein has been identified as an estrogen/ERα-regulated gene in the pituitary. The findings we may make from the study of Amn function in the pituitary will add to the understanding of the molecular mechanisms of estrogen priming of the pituitary in preparation for the LH surge. Supported by a COBRE grant from NIH NCRR: P20 RR15592. (poster)

Amnesiac regulates sleep onset and maintenance in Drosophila melanogaster

The adenylate cyclase/cAMP signaling pathway and adult mushroom bodies (MBs) have been shown to play an important role in sleep regulation in Drosophila. The amnesiac ( amn) gene, encodes a neuropeptide that is homologous with vertebrate pituitary adenylate cyclase-activating peptide (PACAP), is expressed in dorsal paired medial (DPM) neurons and is required for the middle-term memory (MTM) in flies. However, the role of amn on regulation of sleep is as yet unknown. Here we provide evidence that amn plays a major role on sleep maintenance and onset in Drosophila. Flies with the amnesiac allele, loss-of-function amn X8 mutation, showed a fragmented sleep pattern and short sleep latency. Moreover, homeostatic regulation was disrupted in amn X8 mutants after sleep deprivation. Sleep maintenance was also influenced by disruption of neurotransmission in DPM neurons with increased sleep bout number and decreased sleep bout length. Furthermore, age-related sleep fragmentation and initiation were inhibited in amn X8 mutant flies. These data suggest that amn is required in initiation and maintenance of sleep.

Amnionless (AMN) mutations in Imerslund–Gräsbeck syndrome may be associated with disturbed vitamin B12 transport into the CNS

Familial selective vitamin B12 (cobalamin, Cbl) malabsorption (Imerslund-Gräsbeck syndrome, IGS, OMIM 261100) is a group of autosomal recessive disorders characterized by selective malabsorption of Cbl from the terminal ileum in the presence of normal histology. Mutations in the amnionless (AMN) and cubilin (CUBN) genes are known to be causes of IGS. Their gene products combine to form a receptor complex (cubam), which is instrumental in the binding and transport of Cbl in the gut. As opposed to Cbl transport in the terminal ileum, normal transport of Cbl into the CNS is poorly understood and little is known regarding its molecular basis. Studies in adults with neuropsychiatric disease have suggested the presence of an active transport mechanism into the central nervous system constituting a blood-brain barrier (BBB) for Cbl. A child with IGS, compound heterozygous for a missense and a nonsense mutation in the amnionless (AMN) protein gene, was noted to have a high daily cobalamin (Cbl) requirement for neuropsychiatric, but not for systemic metabolic and haematological, remission. Measurements of CSF Cbl revealed evidence that the transport of Cbl into the central nervous system was impaired, and a standard Schilling test was consistent with a dose response of cobalamin transport across the terminal ileum. Amnionless protein is known to be expressed in the fetal and postnatal central nervous system, and is known to be involved in Cbl transport in other tissues such as kidney as well as the gut. It is possible that an active Cbl transport mechanism at the BBB exists, and that the amnionless (AMN) protein may be part of this mechanism, as it is in cobalamin transport in the terminal ileum.

Imerslund-Gräsbeck syndrome in a 15-year-old German girl caused by compound heterozygous mutations in CUBN

Imerslund-Gräsbeck syndrome (IGS) is a recessive disorder of intestinal cobalamin (Cbl) absorption and renal tubular protein reabsorption sometimes accompanied by urinary tract malformation. Mutations in the cubilin (CUBN) and amnionless (AMN) genes have been described as causal defects. CUBN and AMN proteins form the cubam complex that functions as the receptor for the intrinsic factor-Cbl (IF-Cbl) complex in the ileum and for proteins found in the primary urine in the kidney. We report the case of a 15-year-old German girl who presented with megaloblastic anaemia and funicular myelosis due to Cbl-deficiency and selective proteinuria. We clinically diagnosed- and for the first time in a patient of German ancestry-genetically confirmed IGS by detecting a compound heterozygous gene deletion and missense mutation in the CUBN gene. In conclusion IGS should be considered in paediatric patients presenting with symptoms like megaloblastic anaemia, funicular myelosis and benign proteinuria. Diagnosis should be confirmed genetically to avoid further invasive diagnostics, administer proper lifelong treatment and offer genetic counselling.

Genetic heterogeneity of megaloblastic anaemia type 1 in Tunisian patients

Megaloblastic anaemia 1 (MGA1) is a rare autosomal recessive condition characterized by selective intestinal vitamin B12 malabsorption and proteinuria. More than 200 MGA1 patients have been identified worldwide, but the disease is relatively prevalent in Finland, Norway and several Eastern Mediterranean regions. MGA1 is genetically heterogeneous and can be caused by mutations in either the cubilin (CUBN) or the amnionless (AMN) gene. In the present study we investigated the molecular defect underlying MGA1 in nine Tunisian patients belonging to six unrelated consanguineous families. Haplotype and linkage analyses, using microsatellite markers surrounding both CUBN and AMN genes, indicated that four out of the six families were likely to be linked to the CUBN gene. Patients from these families were screened for the Finnish, Mediterranean and Arabian mutations already published. None of the screened mutations could be detected in our population. One family showed a linkage to AMN gene. Direct screening of the AMN gene allowed the identification of the c.208-2A>G mutation, previously described in a Jewish Israeli patient of Tunisian origin and in Turkish patients. This suggests that the c.208-2A>G mutation may derive from a single Mediterranean founder ancestor. For the last family, haplotype analysis excluded both CUBN and AMN genes, suggesting the existence of a third locus that may cause MGA1.

Imerslund-Grasbeck Syndrome Associated With Recurrent Aphthous Stomatitis and Defective Neutrophil Function

Vitamin B(12) deficiency is a well-known cause of recurrent aphthous stomatitis (RAS). However, the mechanism by which this deficiency causes the stomatitis is not well understood. Imerslund-Grasbeck syndrome (IGS) causes vitamin B(12) deficiency and proteinuria due to a defect in the vitamin B(12) receptor. We sought to determine whether the RAS observed in IGS patients is associated with neutrophil dysfunction. We report 3 infants with vitamin B(12) deficiency due to IGS, who presented with borderline or normal hemoglobin concentrations, RAS, and a neutrophil function defect. All 3 patients were homozygous for a splice site mutation affecting exon 4 of the AMN gene. A direct correlation was observed between low serum vitamin B12 levels and defective neutrophil function (low chemotaxis and elevated superoxide production) in the patients. Vitamin B(12) therapy led to an immediate resolution of aphthous stomatitis and full correction of neutrophil function. We demonstrated that serum vitamin B(12) deficiency is associated with a neutrophil chemotactic defect and RAS in IGS patients. We suggest that the RAS observed in these patients is due to this defect.