Factsheet Megaloblastic anemia panel

Megaloblastic anemia panel

Centogene ID: 5074

Gene(s) name (OMIM, HGNC): AMN, CUBN, GIF
Gene OMIM: 605799, 602997, 609342
Panel name Megaloblastic anemia panel
Gene location: 14q32.32, 10p13, 11q12.1

1. Material
minimum DNA (g) minimum EDTA blood (ml) minimum filtercards (pcs)
6 2 2

2. Turnaround time

full gene sequencing

(working days)

20

3. Inheritance pattern
Autosomal recessive

4. Clinical features

Megaloblastosis and megaloblastic anemia represents a heterogeneous group of disorders that

share common morphologic characteristics: large bone marrow and blood cells with an arrest
in nuclear maturation. Nuclear maturation is immature relative to cytoplasmic maturity.
These megaloblasts are the result of the abnormalities in DNA synthesis and, to a lesser
extent, RNA and protein synthesis. Megaloblastic changes are most apparent in rapidly
dividing cells such as blood cells and gastrointestinal cells. Changes are not limited to red
blood cells, as hypersegmented neutrophils can be seen on peripheral smears and giant bands
occur in bone marrow. Megaloblastosis can be associated with severe anemia and
pancytopenia, gastrointestinal dysfunction and glossitis, personality changes, psychosis, and
neurological disorders, and they also appear in HIV infections and myelodysplastic disorders
as a result of interference of DNA synthesis. Unbalanced cell growth and impaired cell division
occur, as nuclear maturation is arrested.

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In addition to genetic changes, the most common causes of megaloblastosis are vitamin B-12
(cobalamin) and folate deficiencies, medications, and direct interference of DNA synthesis by
HIV infections and myelodysplastic disorders.

Clinical findings that are characteristic of vitamin B12 deficiency and subsequent megaloblastic anemia
include the following:

- Evidence for achlorhydria such as abdominal discomfort, reflux, early satiety, and
abdominal bloating
- Pernicious anemia or other autoimmune disorders such as thyroid disorders, type I
diabetes, or Addison disease;
- Family history of a gastrectomy
- Conditions that affect the terminal ileum (site of cobalamin absorption), such as
inflammatory bowel disease, sprue, or ileal resection
- Zollinger-Ellison syndrome or pancreatic insufficiency
- Strict vegetarian diet with no consumption of eggs and dairy products.

Additional causes of Vitamin B12-deficiency-caused megaloblastic anemia also include:

- Poor nutrition, alternative diets, and excessive heating and dilution of foods
- Chronic alcoholism
- Conditions that interfere with folate absorption, including inflammatory bowel
disease, sprue or gluten sensitivity, and amyloidosis
- Conditions that increase folate consumption, such as pregnancy, lactation,
hemolytic anemia, hyperthyroidism, and exfoliative dermatitis
- Hyperalimentation and hemodialysis
- Medications that affect folate
- Hereditary disorder: A lifelong history of megaloblastosis or folate deficiency
would suggest a hereditary disorder as the cause.

Mutations in the genes AMN, CUBN and GIF cause hereditary megaloblastic anemia.

The AMN gene encodes the protein amnionless, homologue of mouse AMN protein, a type I
transmembrane protein that is expressed exclusively in the extraembryonic visceral endoderm
layer during gastrulation. AMN recognizes intrinsic factorcobalamin (vitamin B12) and thus
influences its function and can give rise to megaloblastic anemia. The most common
mutations in the AMN gene have been reported in the Norwegian population, and to some
extent in the Finnish population. The AMN gene emerged as a clear candidate for
megaloblastic anemia type 1 because of its high expression in the kidney. Some examples of
AMN mutations include deletion of nucleotide 14 in exon 1 (14delG) of the AMN gene in 3
Norwegian families with recessive hereditary megaloblastic anemia. Additional families in
Norway and other ethnical groups were reported in association with following AMN gene
mutations:

- C-to-T transition at nucleotide 122 in exon 2 (122C-T) in the Norwegian

population
- Replacement of CAG to CGG in the acceptor splice site of intron 3 (208-2A-G),
resulting in skipping the entire exon 4, in the Tunisian Jewish population
- Mutation c.208-2A-G in the Tunisian population.

There are no hotspots regions or mutations for the AMN gene that can be offered, but full
gene sequencing and del/dupl analysis can be used to identify almost 100% of possible
disease-causing mutations. Centogene offers an AMN single gene test as well as the
Megaloblastic anemia panel that includes this gene.

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Another gene related to megaloblastic anemia (MGA1) is the CUNB gene, encoding for cubilin
protein, an intrinsic factor-cobalamin receptor. This protein is closely related to Vitamin B12
function and metabolism and thus it can cause megaloblastic anemia. The most common
mutations in the CUBN gene have been reported in the Finish population, and this is the
reason why CUBN-caused megaloblastic anemia is called megaloblastic anemia of Finish
type.

MGA1 occurs worldwide, but its prevalence is higher in several Middle Eastern countries and
in Norway, and highest in Finland (0.8 in 100,000).

In 16 of 17 Finnish families segregating megaloblastic anemia-1 a 3916C-T transition in the

CUBN gene, resulting in a P1297L substitution in cubilin, was identified. Furthermore, a frame
insertion IVS6, C-G was identified in a Finish family with typical MCA1. Interestingly, a patient
was identified with a novel homozygous G-to-T transversion at the conserved donor splice site
of exon 23 of the CUBN gene. This patient suffered from Imerslund-Grasbeck syndrome, a
subtype of megaloblastic anemia characterized by enterocyte cobalamin malabsorption and
absence of cobalamin intristic factor.

The GIF gene, encoding gastric intrinsic factor, involved in vitamin B12 (cobalamin)
metabolism and resulting in cobalamin deficiency and megaloblastic anemia. The most
common mutations in the GIF gene include the following:

- 68A-G, in codon 5 of the GIF gene, changing the mature protein from glutamine
to arginine at residue 5. In 2 German and Spanish control populations, the
frequency of the change from A to G at nucleotide 68 was 0.067 and 0.038,
respectively
- 4-bp deletion (c183_186delGAAT) spanning positions 104 to 107 in exon 2 of the
GIF gene as the basis of inherited intrinsic factor deficiency
- G-A transition at -1 in the acceptor splice site of intron 1 of the GIF gene was
identified in Kuwaiti families
- homozygosity for a 137C-T transition in exon 2 of the GIF gene, replacing the
conserved serine residue 46 with a leucine (S46L), identified in Turkish families,
etc.

There is no cure for megaloblastic anemia, but replacement treatment with vitamin B12
(cobalamin) could significantly improve the health situation in patients. Proper diet, different
pharmacotherapies, and in some cases transfusions are all therapeutic approaches. Several
clinical trials are also in progress, examining new therapeutic approaches for megaloblastic
anemia.

Centogene offers a Megaloblastic anemia NGS panel that includes the following genes: ATM,
CUBN and GIF.

Each gene in the panel can also be ordered individually as a single gene test.

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5. Differential diagnosis
The differential diagnosis of Megaloblastic anemia-related disorders includes the following
diseases (depending on the major presenting symptoms):

- Neoplasia, acute leukemia, or myelodysplasia

- Nijmegen breakage syndrome
- Folate deficiencies
- Liver disease
- Hypothyroidism
- Hemolytic anemia.

6. Diagnostic strategy
To confirm or establish the diagnosis, we offer full gene sequencing and deletion/duplication
testing with the Megaloblastic anemia NGS panel.

In summary, Centogene offers the following testing strategy to screen for mutations:

Step 1: Megaloblastic anemia panel full gene sequencing bi-directional, covers the
entire coding region, exon/intron boundaries and 200 bp of the gene promoter
of all genes included in the panel, with appropriate Sanger confirmation
Step 2: Megaloblastic anemia panel deletion/duplication testing - covers large
deletions and duplications that cannot be detected by full gene sequencing in
the ATM, CUBN and GIF genes
Step 3: Whole exome sequencing based on NGS technology, with Sanger confirmation of
all mutations/suspected mutations detected essentially covering the coding
region of all known genes (~1% of the human genome).

7. Referral reasons
The following individuals are candidates for this genetic testing:

1. Individuals with clinical symptoms consistent with the disease, with or without a positive
family history (diagnostic testing)
2. Family members with no clinical symptoms who want to know if they are carrying a known
mutation previously identified in an affected relative (carrier testing or predictive testing)
3. Couples who are at risk of having an affected child and wish to pursue pre-implantation
genetic diagnosis (PGD) or prenatal diagnosis (amnio or CVS).

8. Test utility
Confirmation of a clinical diagnosis through genetic testing can allow for genetic counseling
and may direct medical management. Genetic counseling can provide a patient and/or family
members with the natural history of the condition, identify at-risk family members, provide
reproductive risks, explain preconception/prenatal options, and ensure appropriate referral
for patient support and access to information resources.

If several genes are known to cause a particular phenotype, simultaneous testing with NGS
panel may be faster and more cost effective than stepwise testing of individual genes.
Centogenes panels are updated regularly in line with the current literature to ensure
inclusion of all relevant genes. Our team of medical experts offers a free 2nd opinion on the
best testing strategy, so please dont hesitate to contact us at crd@centogene.com and we
will get back to you within 1 working day.

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9. References
- Malek et al. Megaloblastic Anemia. Reference Module in Biomedical Sciences,
from Pathobiology of Human Disease, 2014, Pages 1499-1505.
- Aok AC. Megaloblastic Anemias (Twenty-Fourth Edition), Volume 1, 2012, Pages
1075-1083
- Cortese et al. DNA damage response A double-edged sword in cancer prevention
and cancer therapy. Cancer Letters, Volume 358, Issue 1, 1 March 2015, Pages 8-
16.
- Kook PH. Cobalamin deficiency states: A fine example of the One Medicine
concept. The Veterinary Journal, Volume 196, Issue 2, May 2013, Pages 137-138.
- Rivella et al. Disorders of Iron Metabolism: Iron Deficiency and Iron Overload and
Anemia of Chronic Diseases, Pathobiology of Human Disease, pp 14711487 , 1
September 2014.

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