This document summarizes a case study of a family found to have a novel mutation in the X-linked ALAS2 gene causing sideroblastic anemia. Whole exome sequencing identified a coding mutation in ALAS2. The proband and affected female relatives were found to have skewed X-inactivation favoring the mutant ALAS2 allele, explaining their symptoms. This expands the known mutations and phenotypes caused by ALAS2 deficiencies.

1. Qussay Abbas
‫السورية‬ ‫العربية‬ ‫الجمهورية‬
‫للمتميزين‬ ‫الوطني‬ ‫المركز‬ ‫خريجي‬
‫ّة‬‫ي‬‫الحيو‬ ‫الطبية‬ ‫العلوم‬ ‫برنامج‬

3. The proband
Diagnosis and methods
results
discussion
3-
2-
1-
4-

6. The first delivery
with placenta
accreta with
concomitant blood
loss And RBC
transfusion for the
first time
the proband
delivered healthy
twins ,massive
hemorrhage 2 weeks
after the delivery her
hematocrit to 17%
that required an
additional transfusion

7. A normal BM
karyotype
a macrocytic anemia
and an elevated
serum ferritin level
(800 ng/ml)

8. Laboratory testing of the proband,
her sister and mother
demonstrated RBC macrocytosis.
The brother and daughters of
the proband had normal
complete blood counts.

9. Macrocytic anemia and abnormal dysplastic erythropoiesis: are
features found in a number of congenital and acquired conditions,
including:
Congenital dyserythropoietic anemias (CDAs)
Specific mitochondrial disorders
Some cases lack a clear explanation

11. A. BM aspirate from the proband with erythroid hyperplasia.
B. Prussian blue staining revealed rare erythroblasts with siderotic
granules.
C. BM aspirate with dyserythropoiesis.

12. Core biopsy
demonstrating
erythroid hyperplasia
and dyserythropoiesis.

13. T2* MRI was
performed
estimated liver iron
concentration of 350
μmol, HFE genetic
testing revealed no
mutations
a horseshoe
kidney and a
mildly enlarged
spleen
A trial with the iron
chelator
deferasirox was
attempted
A trial of
phlebotomy with a
target hemoglobin
level of 10 g/dl was
initiated

14. the clinical features
suggested the
possibility of CDA
targeted sequencing of
CDAN1, SEC23B, and
KLF1 was performed
but no mutations were
identified
The proband and family
members then
consented to participate
in a whole-exome
sequencing study
(I-1, II-2, and II-4) had a
total of approximately
200 novel coding and
LOF variants

15. A single notable variant fit the
model of complete penetrance
and was found to be a coding
mutation in the X chromosomal
gene ALAS2.

19. SDS-PAGE gel of WT and mutant ALAS2 Chromatographic purification for both enzymes

21. As only female carriers were found to be
affected ,they reasoned that there may have
been highly skewed X inactivation present in
the individuals in this family.

22. They assessed the X inactivation in whole-blood
genomic DNA by the human androgen receptor
gene polymorphism assay .

23. These values were all within 2 SD of the mean X inactivation
ratios observed in healthy adult females .

24. The proband, who was the most severely affected, had the reatest extent of
skewing toward the mutant allele, at 82.5 % .

26. They noted no change in the intensity of the ALAS2 mutation
by Sanger sequencing.

27. Collectively, these findings suggest that the presence of
some erythroid cells expressing WT ALAS2 in the
patients’ BM was insufficient to compensate for the
presence of erythroid cells expressing the ALAS2
mutation.

29. Most ALAS2 mutation causing sidroblastic anemia are partial
LOF alleles affect heme biosynthesis .
complete ALAS2 LOF in mouse models results in an early
block of erythropoiesis at approximately the pro-
erythroblast or basophilic erythroblast stages.

33. Notes
• As the cells transitioned from the early progenitor stages to
terminally differentiated erythroblasts.
• there was selection against cells expressing the mutant
ALAS2 mRNA, leaving only cells expressing mRNA from the
WT allele

34. Thus, the X inactivation and mRNA
sequencing studies show that :
•

35. 

36. 


39. Macrocytic anemia
Dimorphic Monomorphic
With sufficiently deleterious
ALAS2 mutations
Typically with XLSA

40. Our findings highlight the value of whole-
exome sequencing in diagnostically
challenging cases for the identification of
disease etiology and extension of the known
phenotypic spectrum of disease