Journal of Inherited Metabolic Disease

, Volume 33, Issue 1, pp 17–24

A novel mutation in LMBRD1 causes the cblF defect of vitamin B12 metabolism in a Turkish patient

Authors

  • Susann Gailus
    • Department of General PediatricsMünster University Children’s Hospital
  • Terttu Suormala
    • Metabolic UnitUniversity Children’s Hospital
  • Ayse Gül Malerczyk-Aktas
    • Department of General PediatricsUniversity Hospital of Giessen and Marburg
  • Mohammad R. Toliat
    • Cologne Center for Genomics and Institute for Genetics
  • Tanja Wittkampf
    • Department of General PediatricsMünster University Children’s Hospital
  • Martin Stucki
    • Division of Metabolism and Molecular PediatricsUniversity Children’s Hospital
    • Zürich Center for Integrative Human Physiology (ZIHP)University of Zürich
  • Peter Nürnberg
    • Cologne Center for Genomics and Institute for Genetics
    • Cologne Excellence Cluster on Cellular Stress Response in Aging-associated Diseases (CECAD)University of Cologne
  • Brian Fowler
    • Metabolic UnitUniversity Children’s Hospital
  • Julia B. Hennermann
    • Department of PediatricsCharité University Medical Center
    • Department of General PediatricsMünster University Children’s Hospital
    • Klinik und Poliklinik für Kinder- und JugendmedizinUniversitätsklinikum Münster
Original Article

DOI: 10.1007/s10545-009-9032-7

Cite this article as:
Gailus, S., Suormala, T., Malerczyk-Aktas, A.G. et al. J Inherit Metab Dis (2010) 33: 17. doi:10.1007/s10545-009-9032-7
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Abstract

In the cblF defect of vitamin B12 (cobalamin) metabolism, cobalamin is trapped in lysosomes. Consequently, cobalamin coenzyme synthesis is blocked, and cofactors for methionine synthase and methylmalonyl-coenzyme A (CoA) mutase are deficient. We recently identified LMBRD1 as the causative gene located on chromosome 6q13 and showed that 18 out of 24 alleles in unrelated patients carried the deletion c.1056delG (p.L352fsX18) (Rutsch et al. (Nat Genet 41:234–239, 2009). LMBRD1 encodes the lysosomal membrane protein LMBD1, which presumably facilitates lysosomal cobalamin export. Our patient is the second child of consanguineous Turkish parents. He presented on the second day of life with cerebral seizures due to intraventricular hemorrhage. Plasma homocysteine and urinary methylmalonic acid levels were elevated, and serum cobalamin level was decreased. Synthesis of both cobalamin coenzymes was deficient in cultured skin fibroblasts. The cblF defect was confirmed by somatic complementation analysis. Sequencing of LMBRD1 revealed the novel deletion c.1405delG (p.D469fsX38) on both alleles. Real-time polymerase chain reaction (PCR) revealed reduced messenger RNA (mRNA) levels in patient fibroblasts compared with controls. Transfection of patient fibroblasts with the LMBD1 wild-type complement DNA (cDNA) rescued coenzyme synthesis and function, confirming this new deletion as an additional cause of the cblF defect. This case adds to the spectrum of clinical presentations and mutations of this rare disorder of lysosomal transport.

Abbreviations

AdoCbl

Adenosylcobalamin

LMBRD1, LMBD1

LMBR1 domain containing 1

LMBR

limb region 1

LIMR

lipocalin-interacting membrane receptor

MeCbl

Methylcobalamin

Introduction

Cobalamin (vitamin B12) is an essential water-soluble vitamin that belongs to the family of tetrapyrrole-derived macrocyclic compounds (Kim et al. 2008). It binds a cobalt atom at its center, and the convertible ligand determines its coenzyme function. Cobalamin must be taken up from dietary animal products and is then transported bound to proteins through the human body. Cobalamin, bound to transcobalamin, enters cells by receptor-mediated endocytosis and is shuttled to the cytoplasm by a lysosomal cobalamin transporter. Cobalamin coenzymes are required for only two reactions in the cell: those catalyzed by cytosolic methionine synthase, in the form of methylcobalamin (MeCbl); and mitochondrial methylmalonyl-coenzyme A (CoA) mutase, in the form of adenosylcobalamin (AdoCbl) (Fowler 1998).

There are eight known defects of the intracellular cobalamin pathway (cblA, cblB, cblC, cblD, cblE, cblF, cblG, mut) identified by somatic complementation analysis. All defects are rare inherited disorders associated with either isolated hyperhomocysteinemia (cblD-homocystinuria, cblE, cblG), isolated methylmalonic aciduria (cblA, cblB, cblD-methylmalonic aciduria, mut) or a combination (cblC, cblD-combined, cblF) (Rosenblatt and Cooper 1990; Suormala et al. 2004). All eight cobalamin defects have variable clinical presentations and outcomes (Rutsch et al. 2009).

To date, only 12 patients with a proven cblF defect have been described world wide (Rutsch et al. 2009). The cblF defect is characterized by the inability to transport free cobalamin from the lysosome into the cytoplasm. Thus, after receptor-mediated endocytosis, cobalamin is trapped in the lysosomes and is lacking for synthesis of coenzymes. This leads to hyperhomocysteinemia and methylmalonic aciduria. Patients are often small for gestational age on presentation, develop feeding difficulties in infancy, and show failure to thrive and developmental delay of varying degree from mild to more severe. The cblF defect, as well as the other seven known defects of the intracellular cobalamin pathway, is inherited as an autosomal recessive trait. Recently, the cblF defect has been shown to be caused by mutations of the LMBRD1 gene, which is located on chromosome 6q13. Eighteen of 24 alleles in unrelated patients carried the deletion c.1056delG (p.L352fsX18) in exon 11. LMBRD1 encodes the lysosomal membrane protein LMBD1, the function of which is still not known (Rutsch et al. 2009). Here we report a male Turkish patient with the cblF defect due to a novel homozygous mutation in the LMBRD1 gene.

Case report

The male proband is the second child of first-degree consanguineous Turkish parents. The first-born brother is healthy. The mother experienced two miscarriages in the past. Serial ultrasound scans during pregnancy were normal, and the boy was delivered spontaneously in the 38th week of gestation. He was small for gestational age, weighing 1,790 g (<3rd percentile) and 46 cm (3rd –10th percentile) long at birth. Additionally, he showed microcephaly, with a head circumference of 30.5 cm (<3rd percentile). Histological examination of the placenta revealed ascending chorioamnionitis and reduced placental diffusion capacity. On the second day of life, the boy developed generalized cerebral seizures caused by right-sided intraventricular hemorrhage (IVH) grade III. A dilated eye examination revealed normal fundus and no retinal hemorrhage. He received phenobarbital for the first 2 weeks of life. Propionyl carnitine in dried blood spots was elevated at 7.57 µmol/l (normal ≤4.2 µmol/l) in selective screening, and elevated ratios were obtained: C3/C0: 0.29 (normal ≤0.15); C3/C2: 0.24 (normal ≤0.16); C3/C16: 5.85 (normal ≤5.04). Repeated screening on the fifth day of life revealed similar propionyl carnitine levels and its corresponding ratios. Further metabolic workup in the first week of life revealed hyperhomocysteinemia (plasma homocysteine elevated to 216 µmol/l; normal <10 µmol/l), hypomethioninemia (plasma methionine 15 µmol/l; normal range 20–40 µmol/l), and methylmalonic aciduria (urinary methylmalonic acid at 176 mmol/mol creatinine (normal <1.9 mmol/mol creatinine). Serum cobalamin level of the patient was decreased to 118 ng/l (normal 243 –894 ng/l); serum levels of vitamin B6 (75.7 µg/l) and folate (12.6 µg/l) were within the normal ranges. Blood count, performed on the first day of life, revealed no abnormalities [hemoglobin (Hb) 19.1 g/dl, mean corpuscular volume (MCV) 111 fl, mean corpuscular Hb concentration (MCHC) 38.7 pg]. The mother’s serum cobalamin level and urinary methylmalonic acid concentration were normal. An inborn defect of cobalamin metabolism was suspected, and treatment with hydroxocobalamin was initiated at the age of 8 days. Plasma homocysteine and urinary methylmalonic acid levels became normal within 2 weeks of substitution with 1 mg hydroxocobalamin intravenously per day. Treatment was continued by intramuscular administration of 1 mg hydroxocobalamin fortnightly. Because serum methionine levels did not increase during hydroxocobalamin treatment, oral application of 50 mg/kg day betaine monohydrate was started. Consecutively, serum methionine levels increased to 32 µmol/l. Within the first month of life, while on hydroxocobalamin treatment, microcytic hypochromic anemia developed (Hb 8.0–8.8 g/dl, MCV 68 fl, MCH 24 pg). A blood smear revealed no abnormalities, and iron deficiency was excluded (serum iron 7.3 µmol/l; serum ferritin 30 µg/l).

The boy started to walk independently and to use words at the age of 18 months. At the time of writing this paper, at the age of 26 months, his psychomotor developmental status, tested according to the Bailey Scales of Infant Development (Bailey and Bricker 1986), was 3 standard deviations (SD) below average; height was between the third and tenth percentiles, but weight and head circumference were still below the third percentile. On long-term treatment with hydroxocobalamin and betaine monohydrate, the levels of homocysteine in plasma, methionine in plasma, propionyl carnitine in dried blood spots, and methylmalonic acid in urine remain completely normal. In contrast, microcytosis and hypochromasia of the red blood cells (RBCs) persist (Hb 12.7 g/dl, MCV 72 fl, MCH 24 pg), whereas hemolysis was ruled out by normal levels for lactic dehydrogenase (LDH; 257 U/l), haptoglobin (87 mg/dl), and free Hb (6 mg/dl).

Material and methods

A skin biopsy to culture fibroblasts was obtained from the patient after informed consent, and the cell line was tested by somatic complementation analysis, as described earlier (Suormala et al. 2004). Genomic DNA was extracted from blood lymphocytes of the patient and his parents after informed consent using the Qiamp DNA Mini Kit (Quiagen) according to the manufacturer’s instructions. All 16 exons and flanking intronic regions of LMBRD1 were amplified, purified, and sequenced, as described previously (Rutsch et al. 2009). To confirm the mutation by PyrosequencingTM and sequencing of 186 control chromosomes, we used the PSQTM HS96A System (Biotage AG). Primers were designed using Primer SNP Design Version 1.01 software (Supplementary Table 1). LMBD1 expression analysis by semiquantitative real-time polymerase chain reaction (PCR) using the ΔΔCt method (Ariani et al. 2004), and functional studies after transient transfection of LMBD1 wild-type cDNA into immortalized fibroblasts, were performed, as described earlier (Coelho et al. 2008; Rutsch et al. 2009)

Statistical analysis

The unpaired t test (two-tailed) with Welch’s correction for unequal variances was applied for testing statistical significance of data on rescue of function using GraphPad Prism software (version 4). P values <0.05 were considered to indicate statistical significance.

Results

Biochemical studies on the patient’s fibroblasts showed findings consistent with an intracellular defect of cobalamin metabolism (Supplementary Table 2). Thus, there was deficient incorporation of propionate and deficient synthesis of methionine, both becoming virtually normal after supplementation of the culture medium with high concentrations of hydroxocobalamin. In addition, synthesis of both AdoCbl and MeCbl was severely deficient. These findings are common for cblD-combined, cblC, and cblF defects (Suormala et al. 2004; Watkins and Rosenblatt 1986). Somatic complementation analysis confirmed the cblF defect in the patient’s cells (Fig. 1a). Molecular analysis of the LMBRD1 gene in the patient showed a novel deletion in exon 14 on both alleles (c.1405delG, p.D469fsX38). The mutation was confirmed by Pyrosequencing™ and was not present in 186 Caucasian control chromosomes. Both parents carried the deletion mutation on one allele (Supplementary Figure 1).
https://static-content.springer.com/image/art%3A10.1007%2Fs10545-009-9032-7/MediaObjects/10545_2009_9032_Fig1_HTML.gif
Fig. 1

Biochemical and molecular data of cblF disease exemplified in a typical case. a Somatic complementation analysis in fibroblasts of the patient. Patient cells were mixed with cblC, cblD, and cblF reference cell lines and assayed for incorporation of [14C]propionate both without fusion (–PEG) and after fusion (+PEG) with polyethyleneglycol. For more details, see “Material and methods.” Each column represents a single determination from one experiment. b)LMBRD1 messenger RNA (mRNA) expression levels in fibroblasts of the patient. Relative LMBRD1 mRNA expression levels in fibroblasts of healthy controls (C1–C3) and the patient by real-time polymerase chain reaction (PCR). Results reflect mean of three independent determinations. c–f Rescue of the cblF defect by transfection with the wild-type LMBD1 construct. Immortalized fibroblasts of the patient, a healthy subject (Control) and a patient with the cblD-combined defect were transfected with pcDNA3 vector containing the LMBD1 wild-type construct (LMBD1-WT) using electroporation. Transfection with the pcDNA3 vector without insert (vector only) was included to measure background activities. c and d Synthesis of cobalamin coenzymes MeCbl and AdoCbl, respectively, from [57Co]cyanocobalamin; e formation of methionine from [14C]formate; f incorporation of [14C]propionate in transfected fibroblasts. Columns represent the mean and vertical lines the standard deviation (SD) of single determinations from four replicate experiments. g Representation of the human LMBRD1 gene with 16 exons and six mutations known to date. The exons are indicated as black vertical bars. The exon number is indicated above and the size in base pairs below the vertical bars. Between exons, intron sizes are indicated in base pairs. The novel mutation is highlighted in grey

Analysis of the LMBRD1 expression level showed a significant down-regulation of the transcript in the patient’s fibroblasts compared with expression in fibroblasts of healthy controls (Fig. 1b). Transfection of immortalized fibroblasts of the patient with the LMBD1 wild-type cDNA construct rescued synthesis of MeCbl and AdoCbl, with increases from 2% and 6% of the mean control value to 40% and 54% (Fig. 1c, d), respectively, and virtually normalized their function as measured by formation of methionine and incorporation of propionate (Fig. 1e, f) In contrast, transfection of fibroblasts from a cblD-deficient patient or an unaffected control with the LMBD1 wild-type cDNA construct had no effect on these activities.

Discussion

This male Turkish patient is one of 13 patients with a confirmed cblF defect world wide. So far, the cblF defect was diagnosed only in patients originating from Canada, USA, Germany, and The Netherlands (Rutsch et al. 2009); this is the first patient of Turkish origin. Of 12 cblF patients known in the literature, four were reported to be small for gestational age, three developed feeding difficulties in infancy, six showed failure to thrive, and five were reported to show developmental delay varying from mild to more severe (Table 1). These clinical features, which are common in other defects of intracellular cobalamin metabolism, were also present in our patient. Furthermore, in three previously described patients, congenital heart defects were reported, including atrial septal defect, patent ductus arteriosus (Rutsch et al. 2009, case 6), right dominant atrioventricular septal defect with common atrium and double-outlet right ventricle (Rutsch et al. 2009, case 1) and an apical ventricular septal defect (Waggoner et al. 1998). So far it is unclear whether the congenital heart disease seen in these patients is secondary to unlinked disease genes or related to cblF.
Table 1

Summary of the clinical data of 13 patients with cblF disease

Patient

1

2

3

4

5

6

7

8

9

10

11

12

13

Reference

Rosenblatt et al. 1985

Shih et al. 1989

MacDonald et al. 1992

MacDonald et al. 1992

Wong et al. 1992

Waggoner et al 1998

Rutsch et al. 2009 (case 1)

Rutsch et al. 2009 (case 2)

Rutsch et al. 2009 (case 6)

Gailus et al. 2007; Rutsch et al. 2009 (case 9)

Rutsch et al. 2009 (case 11)

Rutsch et al. 2009 (case 12)

This case

LMBRD1 mutation(s)

L352fsX18

L352fsX18

L352fsX18

K281fsX4

L352fsX18

L352fsX18

T172fsX9

L352fsX18

L352fsX18

T134fsX14

L352fsX18

T237X

D469fsX38

L352fsX18

L352fsX18

L352fsX18

L352fsX18

L352fsX18

L352fsX18

T172fsX9

L352fsX18

L352fsX18

L352fsX18

?

L352fsX18

D469fsX38

Age at diagnosisa

3 weeks

18 days

11 years

13 months

8 months

4 months

3 weeks

2 months

4 weeks

3 weeks

10 months

4 weeks

8 days

Elevated C3 on MS/MS

      

+

  

+

 

+

+

Heart defect

     

+

+

 

+

    

Feeding difficulties

+

    

+

   

+

  

+

Failure to thrive

 

+

  

+

+

 

+

 

+

+

 

+

Tooth abnormalities

+

        

+

+

  

Glossitis/ Stomatitis

+

+

+

  

+

   

+

   

Hypotonia

+

  

+

        

(+)

Seizures

+

           

+

Developmental delay

 

+

+

 

+

  

+

   

+

+

Anemia

 

+

+

 

+

  

+

 

+

  

+

Neutropenia

  

+

+

   

+

     

Thrombopenia

  

+

+

         

Serum B12

 

normal

decreased

 

decreased

normal

   

decreased

  

decreased

Pathologic Schilling testa

+

 

+

 

+

    

+

  

Not performed

Additional symptoms

Dextro-cardia

Skin rashes

Arthritis

Hepato-megaly

Recurrent infections

 

Tracheoeso-phageal fistula

Septic shock

  

Gastritis

 

Grade III IVH

aAge at diagnosis refers to the age of the patient when cobalamin treatment was initialized. b In the Schilling test urinary excretion of labeled cobalamin was measured before and after oral application of intrinsic factor. IUGR-intrauterine growth retardation, SGA- small for gestational age, C3- Propionylcarnitine on newborn screening. IVH- intraventricular hemorrhage.

Diagnosis of the cblF defect can be difficult. Symptoms such as small stature for gestational age, feeding difficulties, failure to thrive, and developmental delay are unspecific. Detection of elevated propionyl carnitine in newborn screening may be a first sign of a defect in cobalamin metabolism, but this can also be found in other metabolic defects, including propionic aciduria due to propionyl-CoA carboxylase deficiency or methylmalonic aciduria due to methylmalonyl-CoA mutase deficiency. Propionyl-CoA is converted to methylmalonyl-CoA by propionyl-CoA carboxylase. The AdoCbl-dependent methylmalonyl-CoA mutase converts methylmalonyl-CoA to succinyl-CoA, which enters the tricarboxylic acid cycle. Elevated propionyl carnitine was detected in four of 13 cblF cases on extended newborn screening (Table 1). Propionyl carnitine is not included in routine newborn screening in Germany (German Newborn Screening Directive, www.g-ba.de), but is available in selective tandem mass spectrometry (MS/MS) screening. We hypothesize that the cblF defect could be more easily detected if screening for propionyl carnitine is included in routine newborn screening programs.

In our newborn patient, initial plasma cobalamin levels were low, which has been described in three previous cblF patients on presentation (MacDonald et al. 1992; Wong et al. 1992; Gailus et al. 2007, Table 1). Low plasma cobalamin levels, as well as elevated plasma propionyl carnitine levels, hyperhomocysteinemia, and methylmalonic aciduria are also found in infants born to mothers with nutritional cobalamin deficiency or pernicious anemia (Campbell et al. 2005; Marble et al. 2008). Maternal cobalamin deficiency was ruled out in the infant reported here. Our case and a previous case (Gailus et al. 2007) indicate that increased propionyl carnitine in association with low serum cobalamin levels is not unique to infants with maternal cobalamin deficiency. We conclude that low cobalamin serum levels in infants need to be more thoroughly explored in order not to miss a case of cblF disease.

Rosenblatt and co-workers initially described the cblF defect in 1985 as an inborn error of cobalamin metabolism characterized by accumulation of non-protein-bound cobalamin within lysosomes (Rosenblatt et al. 1985). Cells with the cblF defect are able to take up cobalamin bound to transcobalamin and release it from this complex. However, they are not able to transport cobalamin across the lysosomal membrane into the cytoplasm (Vassiliadis et al. 1991). This inability to shuttle cobalamin across lysosomal membranes may account for the pathological Schilling test (described in four previous cases, Table 1) and for low plasma levels in affected cblF newborn infants (described in four cases including ours, Table 1) This is unique for the cblF defect compared with other cbl complementation groups and may reflect inability to transport cobalamin from intestinal cells into the blood stream as well as across the placenta into the fetal circulation. Our patient was small for gestational age, which has been described previously in four cblF cases (Table 1). Low cobalamin concentrations might cause intrauterine growth retardation in the fetus, which has been demonstrated previously (Rutsch et al. 2009). However, in our case, placental histology revealed signs of reduced diffusion capacity, which might also help to explain short stature and microcephaly of the infant at birth.

Uniquely, our patient presented with grade III IVH on the second day of life after an uneventful pregnancy. Due to two previous miscarriages by the mother, the fetus had been closely monitored during pregnancy, and no signs of a prenatal insult had been acknowledged. The highly elevated homocysteine levels in our patient might have predisposed the child to cerebral venous infarction with subsequent development and/or progression of IVH. This pathogenic mechanism has been discussed for other thrombophilic risk factors, including the Gln506 variant of factor V in newborn infants (Petäjä et al. 2001), and hyperhomocysteinemia has been shown to be associated with stroke in newborn infants in a larger retrospective study (Hogeveen et al. 2002).

Using genome-wide homozygosity mapping, we recently described the association of the LMBRD1 gene with the cblF defect (Rutsch et al. 2009). Five different mutations of the LMBRD1 gene, all leading to frameshift and premature termination of translation, were detected in 12 affected patients (Fig. 1g). The patients were homozygous or compound heterozygous (one allele not identified in one case) for these mutations. LMBRD1 encodes the 61.4-kDa protein, LMBD1, which consists of 540 amino acids. It could be demonstrated that LMBD1 is a membrane protein of the lysosome with a cytoplasmic C terminus and nine transmembrane helices expressed in different organs, including liver tissue (Rutsch et al. 2009; Wang et al. 2005). Furthermore, LMBD1 shares significant homology with two proteins of a membrane protein family (LMBR1 PF04791), i.e., limb region 1 (LMBR1) protein and lipocalin-1-interacting membrane receptor (LIMR) (Clark et al. 2000; Wojnar et al. 2001). LIMR acts as a cell-surface receptor and internalizes two lipocalins, which function as binding proteins and carry hydrophobic molecules (Flower 1996; Wojnar et al. 2003).

The exact function of LMBD1 is not known. However, location in the lysosomal membrane as well as the rescue of MeCbl and AdoCbl synthesis after transfection with the LMBD1 wild-type construct strongly suggest that LMBD1 is involved in the transport of cobalamin from the lysosome into the cytoplasm (Rutsch et al. 2009).

We now report a further deletion mutation, located in exon 14 of the LMBRD1 gene, which was detected on both alleles in a Turkish infant suffering from cblF defect. Correction of the cblF defect after transfection of the patient’s fibroblasts with the wild-type construct provides further evidence that this novel deletion mutation on exon 14 is responsible for the cblF defect. The mutation leads to a frameshift and a premature termination codon. The significant down-regulation of the transcript in patient fibroblasts is similar to that seen in other cblF patients and is consistent with nonsense-mediated decay of the transcript (Rutsch et al. 2009). Thus, the underlying mechanism of all reported mutations so far is likely the same at the level of mRNA. It is of interest in this respect that no other mutations than nonsense mutations have been identified in cblF patients to date, though not all of the mutations in the 12 previously identified patients were identified. Thus, one may hypothesize that LMBRD1 missense mutations might cause a milder phenotype, which might manifest later in life.

The patient responded to parenteral therapy with 1 mg hydroxocobalamin twice a month, as revealed by a normalization of plasma homocysteine and urinary methylmalonic acid levels. Methionine plasma levels normalized after addition of betaine. We conclude that the application of a high amount of cobalamin overcomes the defective lysosomal cobalamin export, perhaps by an alternative transport mechanism. This is different compared with other cbl complementation groups, such as cblC, where much higher doses given more frequently are often ineffective in correcting the biochemical defect. However, the effect of cobalamin storage within lysosomes is not known, and one may speculate that this may have some detrimental effect by affecting the function of other lysosomal enzymes. This might explain failure to thrive in affected patients despite cobalamin treatment. However, to date, there is no evidence of cobalamin storage in lysosomes in treated patients with cblF disease. Furthermore, we do not know whether cobalamin will sufficiently cross the blood–brain barrier to correct brain metabolism. It seems likely that lysosomal cobalamin export may also be involved in cobalamin transport across the endothelial cells and into brain cells. This may be corrected ineffectively by parenteral hydroxocobalamin therapy, which could be the reason for the striking developmental delay. Further studies in animal models should address these hypotheses.

In summary, we describe a Turkish patient with the cblF defect of cobalamin metabolism who presented with grade III intraventricular hemorrhage and low serum cobalamin levels in the neonatal period and who carries a novel deletion in the LMBRD1 gene on both alleles, leading to disruption of a putative lysosomal cobalamin exporter. Symptoms of this rare inborn error of metabolism are nonspecific, and diagnosis can easily be missed. To pinpoint the diagnosis, biochemical studies and complementation analysis of patient fibroblasts are indispensable. Treatment consists of parenteral application of hydroxocobalamin, which leads to correction of the biochemical phenotype. However, the long-term effect of cobalamin treatment in cblF patients on lysosomal function and patients’ growth and development remains to be elucidated.

Acknowledgments

The authors thank Ulrike Botschen for expert technical assistance and Torsten Stölting for graphical assistance. SG and FR are supported by funds from “Innovative Medical Research”, Münster University, Germany. FR is supported by the Interdisciplinary Center for Clinical Research, Münster, Germany. TS, MS, and BF are supported by a grant from the Swiss National Foundation (320000-122568/1).

Supplementary material

10545_2009_9032_Fig1_ESM.gif (48 kb)
Supplementary Table 1(GIF 48 kb)
10545_2009_9032_Fig2_ESM.gif (152 kb)
Supplementary Table 2(GIF 151 kb)
10545_2009_9032_Fig3_ESM.doc (139 kb)
Supplementary Figure 1(DOC 139 kb)

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