Identification of a 4-base deletion in the gene in inherited intrinsic factor deficiency

Malabsorption of cobalamin (Cbl) can result from a number of causes such as acquired abnormalities of the stomach,¹  in a familial disorder involving translocation of receptor-bound IF-Cbl,²  and deficiency of transcobalamin II (TCII), because this protein transfers the absorbed Cbl into the blood.³  The most common cause of Cbl malabsorption is pernicious anemia (PA) with atrophic gastritis, resulting in achlorhydria and reduced IF secretion.⁴  Patients have been described with juvenile PA,⁵  childhood familial malabsorption of Cbl,⁶  and juvenile congenital PA.⁷  The cloning of the gene for IF and localization to chromosome 11q13^8,9  provide the molecular probes to identify defects involving the IF gene.

Inherited IF deficiency is a rare form of Cbl malabsorption in which the gastric mucosa as well as acid and pepsin secretion is normal, but selective malabsorption of Cbl occurs because of lack of functional IF protein.¹⁰  The disorder presents in early childhood, and these patients invariably lack autoantibodies to IF or parietal cells.¹¹  Many variants of IF deficiency have been described, including an unstable IF protein that is susceptible to acid and trypsin,¹²  an IF with lower affinity for Cbl and the ileal receptor,¹³  and an IF protein that binds Cbl but does not bind to the receptor.¹⁴  The genetic basis for the lack of a functional IF has not been defined in these patients.

The proband was the first child of a 15-year-old mother of African-American origin. The history provided by the mother indicated that the father was 18 years old and of the same racial origin. The child was initially evaluated for severe anemia at age 3 years. Bone marrow showed megaloblastic erythroid hyperplasia. On admission, she received packed red blood cells and was treated with 100 μg B₁₂ injections weekly. At age 11, the patient was admitted to the hospital for recurrence of anemia because of discontinuation of therapy. She was treated with weekly vitamin B₁₂ injections (1000 μg), and her hemoglobin increased to 13.3 g/dL (133 g/L). The hematologic findings from both admissions are summarized in Table 1. A Schilling test¹⁵  established malabsorption of B₁₂ (0.8% of the dose excreted in 24 hours) which was corrected to 11% of the dose by coadministration of IF. Her serum lacked anti–intrinsic factor autoantibodies. Informed consent according to the Declaration of Helsinki was obtained from the patient and the mother for additional studies to identify the cause of the disorder.

Gastric juice was collected following pentagastrin stimulation, pepsin was inactivated by raising the pH to 10, and, after lowering to pH 7, stored at –20°C. The Cbl-binding capacity in the gastric juice was measured by a competitive binding assay,¹⁶  and the specific binding of Cbl by IF was determined by preincubating the gastric juice with cobinamide (Cbi) to saturate the haptocorrin (HC).¹⁷  Cbl-binding proteins in the gastric juice were also analyzed by gel filtration chromatography of protein bound [⁵⁷Co]Cbl and by immunoprecipitation of protein bound [⁵⁷Co]Cbl by using antiserums specific for IF and for HC, generated in our laboratory. These antiserums were also used to quantify immunoreactive IF and HC in the gastric juice by using radioimmunoassay (RIA) protocols similar to that described for measuring TCII in serum.¹⁸ 

Genomic DNA was isolated from the peripheral blood of the patient and her mother. Exons encoding the IF protein were amplified by the polymerase chain reaction (PCR) by using primer pairs corresponding to the intronic regions flanking each exon.⁸  Genomic DNA (1 μg), deoxynucleotide triphosphate (dNTP) nucleotides (0.2 mM each), 2.5 U Taq polymerase, and 10 pmol forward and reverse primers in a volume of 50 μL were subjected to 30 cycles of denaturation, annealing, and extension at 94°C, 58°C, and 72°C, respectively, each for 1 minute. The PCR product was separated in a 2% agarose gel, the amplified fragment was excised, and the DNA was recovered. The nucleotide sequence of each amplified fragment was determined by the dideoxy chain termination reaction.¹⁹ 

The onset of megaloblastic anemia at an early age with IF lacking in the gastric juice and normal acid output following pentagastrin administration (Table 2), is characteristic of hereditary IF deficiency. An abnormal Schilling test that was corrected by coadministration of IF provided additional evidence that the Cbl malabsorption was due to lack of IF. The inhibition of Cbl binding in the gastric juice by Cbi indicated absence of IF in the patient's gastric juice (Table 2), and this inhibition was confirmed by the absence of a [⁵⁷Co]Cbl-labeled protein peak corresponding to IF by size exclusion chromatography (Figure 1A). The protein peak observed with the patient's gastric juice was identified as HC by its larger size and by immunoprecipitation of the [⁵⁷Co]Cbl-labeled protein with an antiserum to HC (Figure 1B). There was no immunoreactive IF protein in the gastric juice by RIA using a monospecific polyclonal antiserum to human IF. With the exception of functionally abnormal IF proteins that have been identified,^12-14  most patients either lack an intact protein or have a partially degraded IF protein.²⁰ 

Analysis of exons amplified by PCR showed normal nucleotide sequences except for exon 2. There was a 4-base deletion spanning nucleotides 104 to 107 (GAAT) in exon 2 (Figure 2A, upper panel), generating a frame shift in the codons and premature termination of translation 20 bases downstream (Figure 2B). This sequence predicts a translated product of 67 amino acids of which the last 7 are changed and must be unstable because no immunoreactive IF was detected in the gastric juice. One normal allele and one mutated allele were evident in the mother's DNA from the overlapping nucleotide peaks in the sequence immediately 3′ of the deletion (Figure 2A, lower panel). The PCR product from the mother's genomic DNA showed heteroduplex formation, appearing as a doublet in the agarose gel (Figure 2C) which is characteristic of insertions or deletions. Digestion of this PCR product with BstXI or with BsaBI generated an approximate 200-bp fragment (Figure 2C), confirming the presence of one normal and one mutated allele. The presence of the uncut fragment and low yield of the cut fragment with these enzymes were due to inefficient cutting by both enzymes which require unique conditions and temperatures of 55°C and 60°C, respectively, for activity. Because exon 2 contained only the mutated sequence, the child could be either homozygous or hemizygous. The latter seems unlikely, because Southern blot analysis of genomic DNA digested with HindIII, HinfI, and PstI that cut 3 or more times in the gene showed banding patterns and hybridization signals similar to those observed with control DNA. Moreover, the yield of the PCR product for each of the exons was also similar to that obtained for the control, suggesting the presence of both alleles in the propositus. Consanguinity could not be established because of lack of family history and DNA from the father. However, on the basis of available data, it seems likely that the patient is homozygous for the deletion and the inheritance is autosomal recessive.