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Exome Sequencing Identifies MPL As A Causative Gen
Exome Sequencing Identifies MPL As A Causative Gen
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1
Centre for Paediatrics, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University
of London, London, UK; 2University College London Genetics Institute, Gower Place, London, UK; and 3St Mary’s Hospital
Paddington, London, UK
ABSTRACT
The primary cause of aplastic anemia remains unknown in patients. This study shows for the first time a link between
many patients. The aim of this study was to clarify the genetic homozygous MPL mutations and familial aplastic anemia. It
cause of familial aplastic anemia. Genomic DNA of an affected also highlights the important role of MPL in trilineage
individual from a multiplex consanguineous family was hematopoiesis.
hybridized to a Nimblegen exome library before being
sequenced on a GAIIx genome analyzer. Once the disease Key words: aplastic anemia, MPL, exome sequencing.
causing homozygous mutation had been confirmed in the
consanguineous family, this gene was then analyzed for muta- Citation: Walne AJ, Dokal A, Plagnol V, Beswick R, Kirwan M,
tion in 33 uncharacterized index cases of aplastic anemia (<13 de la Fuente J, Vulliamy T, and Dokal I. Exome sequencing iden-
years) using denaturing HPLC. Abnormal traces were con- tifies MPL as a causative gene in familial aplastic anemia.
firmed by direct sequencing. Exome sequencing identified a Haematologica 2012;97(4):524-528.
novel homozygous nonsense mutation in the thrombopoietin doi:10.3324/haematol.2011.052787
receptor gene MPL. An additional novel homozygous MPL
mutation was identified in the screen of 33 aplastic anemia ©2012 Ferrata Storti Foundation. This is an open-access paper.
2 affected children (Family 1: 4-I and 4-III) and where iden- Telomere length measurement
tical homozygous markers were identified, all family mem- Telomere lengths were measured using a multiplex real
bers were typed at this and flanking markers. Areas of time PCR method as described previously.9-10 T/S ratios
homozygosity which were consistent with linkage were obtained for the patient samples were compared with
noted. healthy control samples and also patients with known
TERC mutations as examples of short telomeres.
Exome sequencing
Approximately 180,000 coding exons from 5µg of Results and Discussion
genomic DNA from patient 4-III (Family 1) were sequenced
on the Illumina Genome Analyser IIx after enrichment Following microsatellite analysis, several regions of
using the NimblGen SeqCap EZ exome library. Sequencing homozygosity shared by the 2 affected individuals were
data were processed through the Illumina pipeline and identified (Family 1, Online Supplementary Figure S1). Given
unique homozygous changes identified by filtering the the large number of genes in these intervals, DNA from
resultant data set against variations reported on dbSNP patient 4-III (Figure 1A) underwent exome sequencing. We
(www.ncbi.nlm.nih.gov/snp/), the 1000 genome project filtered the resultant data set against: i) known variations in
(www.1000genomes.org/) and an in-house data set. published databases; ii) an in-house database; iii) excluding
any changes not in a homozygous interval; iv) based on
MPL mutation detection and gene screening gene function; and v) precedence of a disease with similar
Primers were designed to all coding exons (Online clinical features being caused by mutations in the same
Supplementary Table S1). The mutation c.1248G>A gene. We were able to reduce the number of variations to
p.W416X in exon 8 of MPL, was confirmed by direct study to a homozygous mutation c.1248 G>A,
sequencing using ABI BigDye v3.1. For the other 33 sam- p.Trp416Stop in MPL. MPL (myeloproliferative leukemia
ples, all exons of MPL were screened by denaturing high virus oncogene) encodes the thrombopoietin (TPO) recep-
performance liquid chromatography (dHPLC) using the tor. The interaction of thrombopoietin with its receptor is
Transgenomic Wave DNA fragment analysis system to largely responsible for megakaryopoiesis, platelet activa-
detect heteroduplexes following pair-wise pooling. All tion, as well as the maintenance of hematopoietic stem cells
abnormal elution patterns were sequenced as described (HSC). This also initiates intracellular signaling, including
above. Any coding variations identified were compared to activation of the JAK/STAT, RAS/MAPK and P13K/AKT
dbSNP and 1000 genome databases to determine that they pathways.11 Clinically, biallelic constitutional mutations in
had not been described as a rare variation. MPL have been described in congenital amegakaryocytic
Table 1. Clinical features of the patients with biallelic MPL mutations and a comparison with features of CAMT.
Feature CAMT * Family 1 Family 2
Ethnic origin Tunisia Pakistan
Relationship of parents Consanguineous Non consanguineous but parents
from same village
Age at presentation Typically at birth/infancy 4 years (index case) 3 years 18 months (index case) 3 years
Presenting feature Reduced/absent Severe aplastic anemia Aplastic anemia Rotavirus infection
megakaryocytes with no other (abnormal blood
and platelets clinical abnormalities count noted) Family study
Blood counts (At age 6 yrs) Thrombocytopenia
Hb (g/dL) 7.1 NA 11.3 only
WBC (¥109/L) 2.5 Low
Platelets (¥109/L) 7 14 60
Retriculocytes (¥109/L) 15
Neutrophils (¥109/L) 0.4 1.56
Exclude inherited Yes FA excluded FA excluded FA excluded FA excluded
forms of
thrombocytopenia
Bone marrow Normal cellularity Hypocellular, Hypocellular Hypocellular, mild Hypocellular
cellularity (initially) no dysmyelopoiesis dyserythropoesis
Other non Minor heart, None None None None
hematologic eye and brain abnormalities
abnormalities reported in some
Treatment Various including Successful HLA Successful HLA Close surveillance Close surveillance
platelet transfusions, identical sib BMT identical sib BMT + supportive care
matched BMT (2 yrs post transplant (engraftment by day 11)
full donor chimerism)
MPL mutations Homozygous or c.1248 G>A p.Trp416Stop c.1180 C>T p. Pro394Ser (homozygous)
compound heterozygous (homozygous)
*Definition from Ballmaier and Germeshausen19; BMT: bone marrow transplantation; FA: Fanconi anemia; Hb: hemoglobin; NA: not available; WBC: white blood cells.
thrombocytopenia (CAMT). Patients with CAMT typically (Figure 1A). We then extended our screen to patients that
present with thrombocytopenia in the first few were reported to have AA in childhood but not necessarily
weeks/months of life.12 Knockout studies in mice for either in the neonatal period. From the clinical information avail-
Mpl or Tpo show a significant decrease in the numbers of able, the diagnosis of CAMT was not considered by the
myeloid, erythroid, megakaryocytic and burst colony form- referring physician and it was not always clear if a throm-
ing units, but apart from a reduction in platelet numbers, no bocytopenic phase preceded the presentation of the AA. A
reduction in other peripheral blood counts was observed. total of 33 patients were selected (mean age at presentation
When progenitor assays are performed on BM from 4.5 years; range 3 months-11 years). All coding exons of
patients with CAMT, there is a significant reduction in the MPL were screened and a novel missense mutation was
clonogenic cells of all three lineages when compared with identified in one patient. This patient (Family 2, from
BM from healthy individuals, suggesting that the thrombo- Pakistan) had a homozygous mutation c.1180 C>T
cytopenia and pancytopenia observed in these patients may p.Pro394Ser. A study of additional family members showed
result from loss of function mutations in MPL.13 that a second affected brother was also homozygous for
Sequencing the c.1248 G>A, p.Trp416Stop variation in all this mutation (Figure 1B). Both mutations identified in this
Family 1 members confirmed this novel MPL mutation seg- study were clustered in exon 8. This exon is not reported to
regated with disease in an autosomal recessive manner be widely mutated, and the majority of published muta-
A B
Figure 1. Genetic evaluation of MPL mutations in aplastic anemia. (A) Segregation of the c.1248G>A MPL mutation in Family 1 as identified
by exome sequencing. Filled shapes: affected individuals; arrow: affected base. (B) The homozygous missense mutation c.1180C>T
p.Pro394Ser segregates with disease in Family 2. Filled shapes: affected individuals; arrow: affected base. (C) Relative locations of the
mutations identified in MPL from this study (marked by arrows) and previously published studies. The exon structure and derived protein
structure with the functional domains are shown in the lower panel. Filled diamonds: nonsense mutations; open diamond: missense muta-
tions; filled triangles: frame shift mutations; open triangle: 7bp deletion; S: splice site mutations; *: recurrent mutation with <5 mutated
alleles reported; †: recurrent mutation with 5-10 mutated alleles reported; ‡: recurrent mutation with >10 mutated alleles reported; SP;
signal peptide; TM; transmembrane domain. Adapted from Ballamaier et al.19
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