Inherited cobalamin malabsorption. Mutations in three genes reveal functional and ethnic patterns

Abstract

Background: Inherited malabsorption of cobalamin (Cbl) causes hematological and neurological abnormalities that can be fatal. Three genes have been implicated in Cbl malabsorption; yet, only about 10% of ~400-500 reported cases have been molecularly studied to date. Recessive mutations in CUBN or AMN cause Imerslund-Gräsbeck Syndrome (IGS), while recessive mutations in GIF cause Intrinsic Factor Deficiency (IFD). IGS and IFD differ in that IGS usually presents with proteinuria, which is not observed in IFD. The genetic heterogeneity and numerous differential diagnoses make clinical assessment difficult.

Methods: We present a large genetic screening study of 154 families or patients with suspected hereditary Cbl malabsorption. Patients and their families have been accrued over a period spanning >12 years. Systematic genetic testing of the three genes CUBN, AMN, and GIF was accomplished using a combination of single strand conformation polymorphism and DNA and RNA sequencing. In addition, six genes that were contenders for a role in inherited Cbl malabsorption were studied in a subset of these patients.

Results: Our results revealed population-specific mutations, mutational hotspots, and functionally distinct regions in the three causal genes. We identified mutations in 126/154 unrelated cases (82%). Fifty-three of 126 cases (42%) were mutated in CUBN, 45/126 (36%) were mutated in AMN, and 28/126 (22%) had mutations in GIF. We found 26 undescribed mutations in CUBN, 19 in AMN, and 7 in GIF for a total of 52 novel defects described herein. We excluded six other candidate genes as culprits and concluded that additional genes might be involved.

Conclusions: Cbl malabsorption is found worldwide and genetically complex. However, our results indicate that population-specific founder mutations are quite common. Consequently, targeted genetic testing has become feasible if ethnic ancestry is considered. These results will facilitate clinical and molecular genetic testing of Cbl malabsorption. Early diagnosis improves the lifelong care required by these patients and prevents potential neurological long-term complications. This study provides the first comprehensive overview of the genetics that underlies the inherited Cbl malabsorption phenotype.

Figure 1

Illustration of the proteins cubilin and amnionless mutated in IGS and intrinsic factor mutated in IFD, leading to inherited cobalamin malabsorption. Mutations that cause IGS in cubilin were restricted to exons 1–28 that encode the amnionless binding domain (EGF1-8) and the IF-Cbl binding region (CUB5-8). Two other mutations located towards the carboxy-terminal end (p.Ser2785fs in CUB20 and p.Ile2984Val in CUB22) caused proteinuria. Mutations in amnionless and intrinsic factor were located throughout the protein and many mutations affect splicing (Table 1). The mutational hotspot in AMN includes the transmembrane domain and flanking GC-rich repetitive genomic sequences that are apparently unstable (see text). CUB means complement C1r/C1s, Uegf, and Bone morphogenic protein-1, EGF means epidermal growth factor repeats, IF means intrinsic factor, CR means cysteine-rich domain, SS means signal sequence (aa 1–19), TM means transmembrane domain (aa 360–380), and aa means amino acid. The proteins are not drawn to scale.

Figure 2

Flow-diagram of the genetic diagnostic strategy in inherited cobalamin malabsorption.

Figure 3

Release of cobalamin from the food and intestinal uptake. Cobalamin (Cbl) uptake and transport pathway from food intake to the portal system. Cbl is released from food proteins (P) by salivary and gastric enzymes and then binds to haptocorrin (H). In the proximal intestine, pancreatic enzymes degrade haptocorrin and Cbl binds to intrinsic factor (IF). In the ileum, the Cbl-IF complex binds to the cubam receptor (CUBN-AMN) and enters the enterocyte (ileal epithelia). Inside the cell, IF is degraded and Cbl is moved to the portal system by an unknown process. The cubam receptor is recycled back to the membrane. In the blood, transcobalamin 2 (TC2) transports Cbl to the tissues. The role of haptocorrin carrying 80% of the Cbl serum fraction is unknown.