Imerslund-Gräsbeck syndrome in a 25-month-old Italian girl caused by a homozygous mutation in AMN
Gianpaolo De Filippo
Dipartimento di Medicina Clinica e Chirurgia, Universita degli Studi Federico II
Servizio di Endocrinologia Pediatrica, AORN Gaetano Rummo
Laboratorio di Patologia Clinica, AORN Gaetano Rummo
Dipartimento di Medicina Clinica e Chirurgia, Universita degli Studi Federico II
Actual address: Service d'Endocrinologie et Diabetologie Pediatrique, Hopitaux Universitaires Paris Sud, Hopital Bicetre
Le Kremlin Bicetre
Istituto di Genetica e Biofisica Adriano Buzzati-Traverso CNR
Imerslund-Grsbeck 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).
Imerslund-Grsbeck syndrome or juvenile megaloblastic
anemia (IGS, OMIM #261100) is a rare autosomal
recessive disorder independently described by Imerslund
 and Grsbaeck  in 1960. It is characterized by
vitamin B12 (cobalamin, cbl) deficiency due to selective
malabsorption of the vitamin and usually results in
megaloblastic anemia appearing in childhood (but not
immediately after birth). It is responsive to parenteral vitamin
B12 therapy. Mild proteinuria is frequently but not
Mutations in the cubilin (CUBN, OMIM # 602997) 
and amnionless (AMN, OMIM # 605799) genes 
account for most cases of IGS, whereas mutations in the
gastric intrinsic factor gene (GIF, OMIM # 609342), which
cause intrinsic factor deficiency (IFD), phenocopy genuine
Approximately 300 IGS cases have been published
worldwide, with new cases predominantly appearing in
eastern Mediterranean countries. The estimated
prevalence (calculated based on Scandinavian data) is less
than 6:1,000,000 . However, many cases may be
IGS patients usually present within the first 5 years of
life with megaloblastic anemia, proteinuria and decreased
serum vitamin B12 levels, which are sometimes
accompanied by neurological symptoms . Failure to-thrive is also
associated with IGS [8,9].
Diagnosing IGS is a time-consuming and often
inconclusive procedure based primarily on excluding other causes
of Cbl deficiency, which are many and vary with age (e.g.,
malnutrition, lack of Intrinsic Factor, consumption of Cbl
by parasitic worms or bacterial overgrowth of the small
intestine, exocrine pancreatic insufficiency, ileitis terminalis,
congenital defects of CUBN, AMN, GIF, TCNI and TCNII,
and pharmacotherapy) .
Until recently, absorption tests with radiocobalt-labeled
cobalamin were routinely used to study patients with
megaloblastic anemia and related conditions. Schillings urinary
excretion technique was the most popular method .
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 the case of a 25-month-old Italian girl who
presented with failure to-thrive and megaloblastic anemia.
She was born at 38 weeks of gestation by cesarean section
from parents of southern Italian origin who were not aware
of any consanguinity. Her birth weight was 2.950 kg, and
length was 49 cm. The perinatal and neonatal periods were
uneventful. She was breast-fed until 6 months and received
only vitamin D supplementation. Her ponderal growth was
normal (at the 50th percentile) until the third month of life,
when it slowly but progressively began to decelerate from
the 50th percentile to the 5th percentile by 21 months. Her
statural growth was consistently at the 25th percentile. Her
psychomotor development was normal for her age.
At 21 months, she began to lose her appetite, and an
obstinate constipation appeared, partially responsive to
an osmotic laxative. Over the course of the next three
months, she lost weight (0.200 kg) and then began
vomiting once daily for the next week.
At admission, she appeared tired and pale. Her weight
was 9.370 kg, length was 85 cm (25th percentile) and
cranial circumference was 49 cm (75th percentile). Her heart
rate was 120 bpm, blood pressure was 80/50 mmHg, and
her temperature was 36.6C. Her chest was normal to
auscultation. Her abdomen was soft, and her liver was not
palpable. Her skinfold was normal with dry skin. There
were no dysmorphic features. The neurological findings
were strictly normal.
The hematological parameters indicated macrocytic
anemia hemoglobin 7.8 g/dl, hematocrit 23.9, RBC 1.99
cells/mcL, MCH 39.2 pg, MCV 120.1 fl, reticulocytes
0.7%, WBC 5,980/mcL, platelets 276.000/mcL and
reduced serum Cbl level (<100 pg/ml, n.v. 211-911) in the
presence of normal serum folate levels (13.2 ng/L). A
peripheric smear showed the presence of neutrophilic
granulocytes with an augmented volume and
hypersegmentated core. All other biochemical indexes were normal
(liver,kidney and thyroid function, inflammatory markers,
malabsorbtion and celiac disease markers). The urinary
excretion of total protein and albumin was normal.
Treatment with parenteral vitamin B12 was initiated
(500 g/day I.M. for 5 days) and resulted in rapid
improvement of symptomatology, with recovery from vomiting
At a three-year follow-up appointment, the girl showed
normal somatic development, with weight and height
at the 50th percentile and normal psychomotor
development on a bimestral administration of vitamin B12
(1,000 g I.M.) regimen. Her hematological parameters
were stably normalized.
To substantiate a clinical diagnosis of IGS, we assessed
the familial history of the index patient. A more accurate
analysis of the genealogic tree revealed the presence of
consanguinity (Figure 1). Thus, after obtaining written
informed consent, we proceeded with genetic testing.
All coding regions including intron/exon boundaries
of the AMN and CUBN genes were analyzed by direct
sequencing. We identified a heterozygous missense
variation, c.1165C > A, in exon 11 of the CUBN gene, which
causes the substitution of threonine for proline at
position 389 of the CUBN protein. This change has been
reported as a polymorphism (rs1801224) in the dbSNP
database  with a minor allele frequency of 0.45.
Mutational analysis of the AMN gene identified the
presence of a previously described mutation (c.208-2
A > G)  in the splice acceptor site of exon 4; the patient
was homozygous for this mutation. Segregation analysis
revealed that the parents were both heterozygous for the
Figure 1 Pedigree of the patient. Cobalamin levels are indicated
(pg/ml; n.v. 211-911). Heterozygous subjects have normal levels
AMN mutation. This change was absent in 600 unrelated
subjects from the same geographical area.
To determine if this mutation results in the skipping
of exon 4, causing aberrant splicing of the transcript, as
previously described , mRNA analysis was performed.
mRNA derived from lymphocytes of the father of the
index patient was reverse transcribed, amplified with
specific primers and sequenced. Two bands were
observed, corresponding to transcripts derived from both
canonical and alternative splicing. In control mRNA,
only the canonical transcript was present.
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.
The condition is rare and if in some cases (i.e. in
patients with mild proteinuria) is easier to suspect IGS, in
some patients the first symptoms could be vague and it
could be more difficult to suspect the disease. Early
diagnosis is important and life-long treatment with vitamin
B12 is necessary.
In ethnic groups in wich there is a low frequency of
consanguineous marriages ad reasonably a lower
prevalence of autosomal recessive disorders as IGS,
diagnosis should be suspected (and thus mutational analysis
performed) only after excluding other common causes of
failure to thrive, general malabsorption and megaloblastic
anemia. The absence of well-known causes of
failureto-thrive, such as recurrent infections or gastrointestinal
complaints, suggests that the metabolic disturbances
caused by an isolated cobalamin decificiency as seen in
IGS can elicit a failure to thrive [8,9].
The CUBN and AMN genes encode the two subunits
(cubilin and amnionless) of the cobalamin-intrinsic factor
of the ileal mucosa . The cubilin-amnionless complex
is called cubam and is considered to be essential for
intestinal cobalamin uptake, renal protein reabsorption and
early rodent embryogenesis .
The reported AMN mutation (a.208-2 A > G) has been
described in people of Sephardic Jewish or Turkish
descent as well as in Arabic families from Jordan. One case
from the USA had Hispanic roots, but, based on the
patients name, the individual was judged to be of Jewish
ancestry, and one case from Spain was without detailed
ethnic information. The occurrence of this particular
mutation in patients originating mainly from the eastern
Mediterranean area suggested that c.208-2A > G may
be a founder mutation. Given that genetic analysis for
newly diagnosed IGS cases is complex, it has been
suggested that AMN c.208-2A > G should be considered first
when dealing with patients originating from Turkey,
Jordan, Spain, or Tunisia or with an ethnic Sephardic
background . The current case represents the first time that
this mutation has been described in a patient of Caucasian
Concerning treatment issues, the vitamin B12 deficiency
is first corrected by giving intramuscular injections of
cobalamin and is recommended that these injections are then
repeated regularly for the rest of the patients life. Several
therapeutic regimens have been proposed: 1000 g of
hydroxycobalamin i.m. daily for 10 days then 1000 g i.m.
once a month  or 1000 g i.m. weekly for 1 month, then
1000 g i.m. every three or six months . Successful
treatment of IGS with 1 mg of vitamin B12, orally
administered at 2-week intervals, has been reported . In view
of the accumulating evidence that subclinical deficiency of
cobalamin may contribute to the development of
atherosclerosis, dementia and osteoporosis [18,19] and
considered that cobalamin is non-toxic, it is suggested that
patients receive a higer dose of cobalamin than necessary,
rather than an insufficient dose . In our case, we decided
to taper the dose according to clinical and laboratory
response and the outcome was strongly favorable on long
term with the administration of 1000 g of vitamin B12 i.
m. every two months.
Our findings indicate that IGS should be considered
when diagnosing children with megaloblastic anemia
and failure to thrive. Diagnosis should be confirmed
genetically, first focusing the study on genes suspected
on the basis of ethnicity and successively, if indicated,
expanding the research to other potentially involved
genes. Genetic confirmation substantiates clinical
findings and the necessity of adequate lifelong therapy.
Written informed consent was obtained from the
patients parents and all studied subjects for publication of
this Case Report and any accompanying images. A copy
of the written consent is available for review by the
Editor-in-Chief of this journal.