Download Exome sequencing to define a genetic signature of plasma cells in

Exome sequencing to define a genetic signature of plasma cells in systemic
AL amyloidosis
Brian Walker1, Eileen Boyle1, Dorota Rowczienio2, Christopher Wardell1, Sajitha
Sachchithanantham2, Anna Baginska2, Shameem A Mahmood2, Julian D Gillmore2,
Helen J Lachmann2, Philip N Hawkins2 , Gareth Morgan1, Faith Davies1
and Ashutosh D Wechalekar2
1Centre
for Myeloma Research, The Institute of Cancer Research, London, UK
for Amyloidosis and Acute Phase Proteins, University College London
Medical School, UK
2Center
Systemic amyloid light chain amyloidosis (AL) is characterized by the deposition of
immunoglobulin light chains as amyloid fibrils in different organs, where they form
toxic protein aggregates. Most AL patients have relatively low levels of circulating
free light chains and bone marrow plasmacytosis. The underlying disease is a
plasma cell disorder, likely a monoclonal gammopathy, but limited data are available
on the biology of the plasma cell clone underlying AL and existing studies have
concentrated on chromosomal abnormalities.
Many of the chromosomal
abnormalities identified in AL are also seen in other plasma cell disorders, such as
monoclonal gammopathy of undetermined significance (MGUS) and myeloma.
These abnormalities include translocations involving the IGH locus, gains of 1q and
deletions of 13q and 17p. Fluorescence in situ hybridization studies have identified
the translocation t(11;14) to be more frequent in AL and hyperdiploidy to be rare.
The causal link between genetic changes in plasma cells and light chain instability
remains unknown and progression to symptomatic myeloma is rare. We report the
initial findings of the first exome sequencing to define the plasma cell signature in AL
and compared this to MGUS and myeloma.
CD138+ cells were selected using either EasySep (Stem Cell Technologies) or
MACSort (Miltenyi) from the bone marrow of 18 AL patients and 5 MGUS patients.
DNA was extracted from the CD138+ cells using the AllPrep kit (Qiagen). Noninvolved DNA was isolated from peripheral white blood cells using the Flexigene kit
(Qiagen). 200 ng DNA was subjected to exome sequencing using NEBNext kit
(NEB) and SureSelect Human All Exon kit v5 and sequenced using 76-bp paired end
reads. Fastq files were aligned to the reference genome using BWA and Stampy
aligners. BAM files were recalibrated using the GATK and deduplicated using
Picard. Paired tumour/normal BAMs were realigned together using the GATK indel
realigner and SNVs were called using Mutect. Copy number data were estimated
using the R package ExomeCNV. The median depth across all samples was 42x
with 97% of the exome covered at 1x and 72% covered at 20x.
Exome data to determine the cytogenetic groups of AL samples identified 42%
hyperdiploid and 21% with t(11;14). The AL samples with t(11;14) did not contain
any other copy number abnormalities. We performed exome sequencing on
samples from patients with MGUS and myeloma to compare the genetic makeup
and mutation spectrum of these well characterised plasma cell neoplasias with AL
samples. MGUS samples had a median of 30 acquired nonsynonymous variants
(range 24-189) and AL amyloidosis samples had a median of 17 acquired
nonsynonymous variants (range 4-44). The AL samples had four recurrent
mutations in PCMTD1 (n=3; L267F, P266S and M187I), C21orf33 (n=2; E72K),
NLRP12 (n=2; L1018P, W959*) and NRAS (n=2; Q61R, Q61H). In this small
dataset, only 5 genes were mutated in both the MGUS and AL samples (DNMBP,
FRG1, HIST1H1B, KRTAP4-11 and MCCC1). In order to assess the similarity (or
differences) of plasma cells in AL to malignant plasma cells in general, we compared
them to a random sampling of 20 multiple myeloma samples which had also been
exome sequenced (median number of acquired nonsynonymous variants = 39 vs. 17
in AL samples). This revealed that that the AL contained 21 mutated genes in
common with the myeloma cohort, including DIS3 and NRAS. There were two DIS3
mutations in one AL sample at c.379D>E (p.D479E) and c.1999A>T (p.M667L), both
of which were in the Ribonuclease II/R catalytic domain. Data on correlation of gene
mutations and organ involvement in AL amyloidosis will be presented.
We conclude that exome sequencing identifies a genetic signature of AL amyloidosis
which is similar to other plasma cell disorders. This not only includes copy number
abnormalities and translocations but also a similar number of nonsynonymous
mutations to MGUS and fewer than the advanced myeloma samples. Study of
further samples is in progress.