Targeted Next-Generation Sequencing Appoints C16orf57 as Clericuzio-Type Poikiloderma with Neutropenia Gene

ArticleinThe American Journal of Human Genetics 86(1):72-6 · December 2009with47 Reads
Impact Factor: 10.93 · DOI: 10.1016/j.ajhg.2009.11.014 · Source: PubMed
Abstract

Next-generation sequencing is a straightforward tool for the identification of disease genes in extended genomic regions. Autozygosity mapping was performed on a five-generation inbred Italian family with three siblings affected with Clericuzio-type poikiloderma with neutropenia (PN [MIM %604173]), a rare autosomal-recessive genodermatosis characterised by poikiloderma, pachyonychia, and chronic neutropenia. The siblings were initially diagnosed as affected with Rothmund-Thomson syndrome (RTS [MIM #268400]), with which PN shows phenotypic overlap. Linkage analysis on all living subjects of the family identified a large 16q region inherited identically by descent (IBD) in all affected family members. Deep sequencing of this 3.4 Mb region previously enriched with array capture revealed a homozygous c.504-2 A>C mismatch in all affected siblings. The mutation destroys the invariant AG acceptor site of intron 4 of the evolutionarily conserved C16orf57 gene. Two distinct deleterious mutations (c.502A>G and c.666_676+1del12) identified in an unrelated PN patient confirmed that the C16orf57 gene is responsible for PN. The function of the predicted C16orf57 gene is unknown, but its product has been shown to be interconnected to RECQL4 protein via SMAD4 proteins. The unravelled clinical and genetic identity of PN allows patients to undergo genetic testing and follow-up.

Full-text

Available from: Ludovica Volpi
REPORT
Targeted Next-Generation Sequencing Appoints C16orf57
as Clericuzio-Type Poikiloderma with Neutropenia Gene
Ludovica Volpi,
1,10
Gaia Roversi,
2,3,10
Elisa Adele Colombo,
3
Nico Leijsten,
4
Daniela Concolino,
5
Andrea Calabria,
6
Maria Antonietta Mencarelli,
7
Michele Fimiani,
8
Fabio Macciardi,
9
Rolph Pfundt,
4
Eric F.P.M. Schoenmakers,
4
and Lidia Larizza
3,
*
Next-generation sequencing is a straightforward tool for
the identification of disease genes in extended genomic
regions. Autozygosity mapping was performed on a five-
generation inbred Italian family with three siblings
affected with Clericuzio-type poikiloderma with neutrope-
nia (PN [MIM %604173]), a rare autosomal-recessive geno-
dermatosis characterised by poikiloderma, pachyonychia,
and chronic neutropenia. The siblings were initially diag-
nosed as affected with Rothmund-Thomson syndrome
(RTS [MIM #268400]), with which PN shows phenotypic
overlap. Linkage analysis on all living subjects of the family
identified a large 16q region inherited identically by
descent (IBD) in all affected family members. Deep
sequencing of this 3.4 Mb region previously enriched
with array capture revealed a homozygous c.504-2 A> C
mismatch in all affected siblings. The mutation destroys
the invariant AG acceptor site of intron 4 of the
evolutionarily conserved C16orf57 gene. Two distinct dele-
terious mutations (c.502A>G and c.666_676þ1del12)
identified in an unrelated PN patient confirmed that the
C16orf57 gene is responsible for PN. The function of the pre-
dicted C16orf57 gene is unknown, but its product has been
shown to be interconnected to RECQL4 protein via SMAD4
proteins. The unravelled clinical and genetic identity of PN
allows patients to undergo genetic testing and follow-up.
PN is an autosomal-recessive hereditary poikiloderma,
a clinically and genetically heterogeneous group of
disorders including RTS. The disorder is characterized by
skin manifestations, mainly a papular erythematous rash
starting on the limbs and face during the first year of life
and evolving into poikiloderma with a pronounced acral
involvement, as well as pachyonychia, especially of the
toenails. One of the most important extracutaneous symp-
toms is an increased susceptibility to infections, mainly
affecting the respiratory system, primarily due to a chronic
neutropenia and to neutrophil functional defects. Bone
marrow abnormalities account for neutropenia and may
evolve into myelodysplasia associated with the risk of
leukemic transformation.
1
PN shows phenotypic overlap with RTS, but a few
specific phenotypic differences point toward a distinct
genetic control. Mutations of RECQL4 (MIM *603780),
the helicase gene mutated in two thirds of RTS patients,
appear to be absent in PN patients.
2,3,4
We genotyped a highly consanguineous Italian family
consisting of 29 subjects across five generations and
including three affected siblings ( Figures 1A and 1B). The
affected members were initially misdiagnosed as RTS
patients as a result of the skin findings that appeared in
all three affected siblings before the first year of life, start-
ing from the face and the extensor surface of the arms
and then evolving into classical poikiloderma. All siblings
also displayed pachyonychia, plantar keratoderma, and
dysmorphisms, especially those related to the midfacial
hypoplasia (Figure 1A). Severe neutropenia due to myelo-
dysplastic hemopoiesis led to recurrent pulmonary infec-
tions, otitis, and sinusitis in two siblings at early infancy.
All patients showed growth retardation, mild spleno-
megaly, and increased level of creatin kinase and lactate
dehydrogenase (LDH). Detailed family description, clinical
findings, neutrophil count, and testing and evolution
of the disease are reported by D.C. (unpublished data).
Concomitant acral poikiloderma, pachyonychia, and
chronic neutropenia, fairly unusual in RTS patients, were
more consistent with the PN diagnosis, which was further
supported by the absence of RECQL4 mutations (data not
shown). All sampled family members provided written
informed consent to participate in the study.
All living subjects (n ¼ 18) were genotyped by a genome-
wide Affymetrix Genechip Human Mapping 262K NspI
SNP Array, and a two-point linkage analysis was performed
with SuperLink (v.1.6), assuming an autosomal-recessive
trait with 100% penetrance.
5,6
SNPs were examined
for informative genomic regions that were homozygous
1
Universita
`
degli Studi di Milano, Dipartimento di Biologia e Genetica per le Scienze Mediche, 20133 Milan, Italy;
2
Unit of Medical Genetics, Fondazione
IRCCS, Istituto Nazionale Tumori, 20133 Milan, Italy;
3
Universita
`
degli Studi di Milano, Genetica Medica, Dipartimento Medicina, Chirurgia e Odontoia-
tria, 20142 Milan, Italy;
4
Department of Human Genetics, Radboud University Nijmegen Medical Center, and Nijmegen Center for Molecular Life Sciences,
P.O. Box 9101, 6500 HB Nijmegen, The Netherlands;
5
Dipartimento di Pediatria, Universita
`
di Catanzaro, 88100 Catanzaro, Italy;
6
National Research
Council, Institute for Biomedical Technologies, 20138 Milan, Italy;
7
Medical Genetics, Department of Molecular Biology, University of Siena, 53100 Siena,
Italy;
8
Clinica Dermatologica Universita
`
di Siena, 53100 Siena, Italy;
9
Universita
`
degli Studi di Milano, Dipartimento di Scienze e Tecnologie Biomediche,
20138 Milan, Italy
10
These authors contributed equally to this work
*Correspondence: lidia.larizza@unimi.it
DOI 10.1016/j.ajhg.2009.11.014. ª2010 by The American Society of Human Genetics. All rights reserved.
The American Journal of Human Genetics 86, 1–5, January 8, 2010 1
Please cite this article in press as: Volpi et al., Targeted Next-Generation Sequencing Appoints C16orf57 as Clericuzio-Type Poikiloderma with
Neutropenia Gene, The American Journal of Human Genetics (2010), doi:10.1016/j.ajhg.2009.11.014
Page 1
2 The American Journal of Human Genetics 86, 1–5, January 8, 2010
Please cite this article in press as: Volpi et al., Targeted Next-Generation Sequencing Appoints C16orf57 as Clericuzio-Type Poikiloderma with
Neutropenia Gene, The American Journal of Human Genetics (2010), doi:10.1016/j.ajhg.2009.11.014
Page 2
in the affected patients but not in any of their healthy
siblings and were checked for quality control after removal
of ambiguous genotypes and data with a call rate under
a 95% threshold.
Linkage analysis identified three regions with a LOD
score > 2.5 (Figure 1C and Table S1, available online),
among which the 16q12.2-q21 region was selected as
the largest genomic interval of which consecutive LOD
score values never fall below 2(Figure 1D). Indeed, the
expected length of the IBD-inherited region around the
disease locus is a function of the inbreeding coefficient of
the proband. The inbreeding coefficient of the last genera-
tion is 3/64, predicting a ~20 cM IBD region.
7
The candidate16q region, spanning 3.4 Mb from SNP-
A_1803188 (rs16954293) to SNP-A_1923765 (rs9939133),
encompasses 276 consecutive SNPs, all consistent with
the predicted inheritance pattern, and contains more
than 80 known and predicted genes (Figure 1E). Such
a list of genes is not manageable in the context of a classical
candidate-gene approach, thus prompting us to proceed
with array capture-mediated next-generation sequencing
(NGS), a strategy enabling an unbiased search for disease-
associated mutations in large genomic intervals.
8
The adopted stepwise procedureis detailed in Figure S1.In
brief, a genomic shotgun library from the 3.4 Mb 16q region
of the siblings was prepared with paired-ends adapters in
accordance with Illumina guidelines. After quality control,
the library was captured by ImaGenes GmbH on a custom
repeats-masked 244K solid array (Agilent).
The target region, dropped from 3.4 Mb to 1.7 Mb with
this procedure, was eventually processed for NGS (Solexa
Technology). ELAND (Illumina GAPipeline 1.0) mapping
carried out against the full Homo sapiens genome yielded
the bed files that were visualized in the UCSC Genome
Browser, build March 2006 (Figure 1E). Mapping with the
Maq program (v.0.7.1)
9
was carried out against the selected
16q region. The best enrichment value (calculated accord-
ing to the formula in Figure S1) was 241 for patient V-2.
The reads were then aligned to the targeted reference
sequence, highlighting a total of 1488 mismatches: 450
were heterozygous mismatches that are unreported in
the UCSC database and are accounted for by their location
within DNA blocks with high sequence homology; i.e., low
copy repeats or duplicons in which chromosome 16 is
enriched (UCSC Genome Browser). As regards the homo-
zygous mismatches, 494 have already been reported and
527 lie within intergenic regions; therefore, we focused
over the remaining 17 unreported regions located within
or very close to genes. We ranked them according to
location and evolutionary conservation. As shown in
Table S2, the A>C SNP position (56.608.737) appeared
to be a strong candidate because it affects the acceptor
splice site of intron 4 of the highly conserved C16orf57
gene (NM_024598.2) mapping to 16q13 (Figure 1E and
Figure 2A); namely, the c.504-2 A> C mutation destroys
the invariant AG dinucleotide splice acceptor (Figure 2B).
Direct capillary sequencing confirmed that the mutation
segregates as expected across the last three generations,
and it also confirmed the carrier (IV-6, IV-7, V-1, V-3) and
noncarrier ( III-4, IV-2, IV-4, IV-5, IV-8) status of all the
living individuals within the pedigree. Subsequent cDNA
analysis on patient V-2 showed an aberrant transcript
106 nucleotides shorter as a result of exon 5 skipping
(Figures 2C and 2D). The predicted protein lacks 35 exon
5-encoded aminoacids and, because of frameshift, differs
from the original protein sequence in the following 61 resi-
dues (p.Thr169IleFsX61) (Figure S2B).
We tested five atypical RTS patients and validated the
association between C16orf57 and PN in the only patient
reported to have the PN clinical hallmark of neutropenia.
Indeed this nonrelated Italian female patient, who was
diagnosed with RTS and myelodysplasia
10
and tested nega-
tive for RECQL4 mutations (data not shown), was found to
be a compound heterozygote for C16orf57 mutations. She
carries a paternally inherited c.666_676þ1del12 mutation
in exon 6 and a maternally inherited c.502A>G missense
mutation in exon 4, (Figure 2E). The c.502A>G mutation
is absent in 175 matched controls and affects a highly
conserved Arginine residue mapping within the conserved
HVSL domain of C16orf57. The comparative aminoacid
sequence analysis in HomoloGene and ClustalW showed
complete conservation at position p.R168 in several
eukaryotic species (Figure S3). cDNA analysis of the com-
pound heterozygous patient resulted in the identification
of two aberrant transcripts (Figures 2F–2I) with the
in-frame skipping of exons 6 (paternal allele) and 4
(maternal allele). Mutated C16orf57 proteins lacking 28
(p.D204_Q231del) and 18 (p.F151_R168del) aminoacids
are predicted (Figures S2C and S2D).
Little is known about C16orf57 or the functions of its
encoded protein, but two independent studies revealed
direct interactions between the C16orf57 and SMAD4
proteins,
11,12
which are interconnected to RECQL4
through HADAC1, TP53, and/or RAD51 (Figure S4). The
phenotypic overlap between RTS and PN can be partially
accounted for by SMAD4-mediated signaling of C16orf57
Figure 1. From Clinical Findings to the Candidate Gene
(A) Affected proband (V-2) and sibling (V-4) at ages 22 and 14, respectively, showing facial poikiloderma extending to buttocks and legs
and pachyonychia of the toes.
(B) Five-generation pedigree: PN patients are indicated by shaded symbols and obligate carriers by dotted symbols. The inbreeding coef-
ficient of the fifth-generation siblings is 3/64.
(C) Genome-wide parametric linkage analysis revealing LOD score > 2.5 on chromosomes 2, 6, and 16 (arrows). See also Table S1.
(D) LOD score diagram magnification of the 16q region (boxed in the upper ideogram): The region spans from SNP A_1803188
(rs16954293 at 16q12.2) to SNP A_1923765 (rs9939133 at 16q21).
(E) Postcapture array coverage of 16q12.2-q21 from V-2 (calculated enrichment value 241) and UCSC Genome Browser view of all genes
mapping to this interval. C16orf57 is circled in red.
The American Journal of Human Genetics 86, 1–5, January 8, 2010 3
Please cite this article in press as: Volpi et al., Targeted Next-Generation Sequencing Appoints C16orf57 as Clericuzio-Type Poikiloderma with
Neutropenia Gene, The American Journal of Human Genetics (2010), doi:10.1016/j.ajhg.2009.11.014
Page 3
to RECQL4. The fact that C16orf57 is significantly ex-
pressed in blood (myeloid lineage) might explain sensi-
tivity to C16orf57 mutations leading to neutropenia and
myelodysplastic features, which are distinctive signs of
PN patients (
1–4
and D.C., unpublished data).
The identification of a gene responsible for PN allows
one to test for the C16orf57 mutation in all RECQL4-nega-
tive RTS patients fitting the PN clinical presentation, who
are probably misdiagnosed, in order to provide adequate
onco-hematological surveillance.
The presumptive role of C16orf57 in myeloid cell matu-
ration and function paves the way for investigation of the
contribution of this gene to both myelodysplasia and
congenital neutropenia syndromes. In addition, the high
degree of evolutionary conservation of C16orf57 increases
the chance that representative animal models can be
developed while such a line of research is pursued.
Supplemental Data
Supplemental Data include four figures and four tables and can be
found with this article online at http://www.ajhg.org.
Acknowledgments
We thank the patients and their relatives for intensive cooperation
in the study; A. Renieri and I. Meloni (University of Siena) for
providing the lymphoblastoid cell line from the sporadic PN
patient; Galliera Genetic Bank for establishing lymphoblastoid
cell lines from the affected siblings of the PN family (Telethon
project GTB07001); and L. Farinelli from Fasteris SA (CH), who
technically and scientifically supported the project steps involving
array capture and next-generation sequencing. Bioinformatics
support for SNP array data was provided by Roland P. Kuiper (Nij-
megen University Centre), and skillful modeling prediction of sig-
nalling pathways was performed by Christian Gilissen (Nijmegen
University Center). As regards human subjects, we followed the
Figure 2. C16orf57 Mutational Analysis
(A) Schematic genomic structure of
C16orf57 (16q13) spanning 20 Kb and 7
exons (dark boxes), encoding a 265 aa
protein. Red arrows point to the identified
mutations (rimers and conditions reported
in Table S3).
(B and E) Pedigrees and sequencing of
C16orf57 genomic mutations: (B) shows
the carrier status of both parents and the
homozygous splicing mutation c.504-
2A>C of the three affected siblings, and
(E) the parental origin and the two muta-
tions c.666_676þ1del12 and c.502A>G
of the compound-heterozygous sporadic
patient. Base changes are indicated with
red characters.
(C, F, and H) Agarose gel electrophoresis,
showing products of different RT-PCR
(primers and conditions reported in Table
S4), on lymphoblastoid cell line RNA.
Lengths of normal and aberrant products
are indicated. The following abbreviations
are used: V-2, homozygous patient; P,
compound-heterozygous patient; Cþ,
normal control; C, negative control; M,
GeneRuler DNA Ladder Mix (Fermentas).
(D, G, and I) Sequence data of the two PN
patients showing aberrant transcripts
due to out-of-frame skipping of exon 5,
in-frame skipping of exon 6 and exon 4,
respectively, and related schematic
diagrams of the mutation-associated mis-
spliced cDNAs.
4 The American Journal of Human Genetics 86, 1–5, January 8, 2010
Please cite this article in press as: Volpi et al., Targeted Next-Generation Sequencing Appoints C16orf57 as Clericuzio-Type Poikiloderma with
Neutropenia Gene, The American Journal of Human Genetics (2010), doi:10.1016/j.ajhg.2009.11.014
Page 4
guidelines of the ethical committee of the University of Milan
(http://www.unimi.it/cataloghi/comitato_etico/CE_Rec_4_2006_
HBMs.pdf). This work was supported by Associazione Italiana per
la Ricerca sul Cancro (grant 2008-2009/4217 to L.L.), CARIPLO
N.O.B.E.L. (project 2007-2009 to L.L.), and Nando Peretti Founda-
tion (grant 2007-2009/14 to L.V.).
Received: September 8, 2009
Revised: November 6, 2009
Accepted: November 17, 2009
Published online: December 10, 2009
Web Resources
The URLs for data presented herein are as follows:
BLAST: Basic Local Alignment Search Tool, http://www.ncbi.nlm.
nih.gov/blast/Blast.cgi
ClustalW software, http://www.hongyu.org/software/clustal.html
ConSeq Server, http://conseq.bioinfo.tau.ac.il
ESE Finder, http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.
cgi?process¼home
Fruitfly, http://www.fruitfly.org/seq_tools/splice.html
Genome-wide Viewer, https://bioinformatics.cancerresearchuk.
org/~cazier01/GWA_View.html
HomoloGene, http://www.ncbi.nlm.nih.gov/homologene
Maq, http://maq.sourceforge.net/
NetGene2 server, http://www.cbs.dtu.dk/services/NetGene2
Illumina, http://illumina.com
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.
nlm.nih.gov/Omim/
Pathway Studio, http://www.ariadnegenomics.com/products/
pathway-studio
Pmut, http://mmb2.pcb.ub.es:8080/PMut
PolyPhen, http://genetics.bwh.harvard.edu/pph
PSIPRED: Protein Structure Prediction Server, http://bioinf.cs.ucl.
ac.uk/psipred
SIFT, http://sift.jcvi.org
STRING, http://string.embl.de
UCSC Genome Browser, http://genome.ucsc.edu; http://genome.
ucsc.edu/cgi-bin/hgTracks
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Please cite this article in press as: Volpi et al., Targeted Next-Generation Sequencing Appoints C16orf57 as Clericuzio-Type Poikiloderma with
Neutropenia Gene, The American Journal of Human Genetics (2010), doi:10.1016/j.ajhg.2009.11.014
Page 5
    • "The function of the 2H domain in UBASH3 is unknown (Tsygankov, 2013). USB1 is linked to a rare hereditary disease, poikiloderma with neutropenia, and functions in the maturation of the U6 snRNA (Colombo et al., 2012; Hilcenko et al., 2013; Volpi et al., 2010; Walne et al., 2010). This enzyme catalyzes the cleavage of the phosphodiester bond between the two terminal nucleotides in the substrate and forms a product with a 2′,3′-cyclic end (Hilcenko et al., 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) is an abundant membrane-associated enzyme within the vertebrate myelin sheath. While the physiological function of CNPase still remains to be characterized in detail, it is known – in addition to its in vitro enzymatic activity – to interact with other proteins, small molecules, and membrane surfaces. From an evolutionary point of view, it can be deduced that CNPase is not restricted to myelin-forming cells or vertebrate tissues. Its evolution has involved gene fusion, addition of other small segments with distinct functions, such as membrane attachment, and possibly loss of function at the polynucleotide kinase-like domain. Currently, it is unclear whether the enzymatic function of the conserved phosphodiesterase domain in vertebrate myelin has a physiological role, or if CNPase could actually function – like many other classical myelin proteins – in a more structural role.
    No preview · Article · Sep 2015 · Brain Research
    0Comments 0Citations
    • "All strains were maintained according to standard laboratory procedures. Lymphoblastoid cell lines ML07136M (P1 in the text) derived from a PN patient and GGB04410M (R1) derived from P1's healthy brother were obtained from Galliera Genetic Bank (Genova, Italy) and were previously described [7] [8]. "
    [Show abstract] [Hide abstract] ABSTRACT: Mpn1 is an exoribonuclease that modifies the spliceosomal small nuclear RNA (snRNA) U6 by trimming its oligouridine tail and introducing a cyclic phosphate group (>p). Mpn1 deficiency induces U6 3' end misprocessing, accelerated U6 decay and pre-mRNA splicing defects. Mutations in the human MPN1 gene are associated with the genodermatosis Clericuzio-type poikiloderma with neutropenia (PN). Here we present the deep sequencing of the >p-containing transcriptomes of mpn1Δ fission yeast and PN cells. While in yeast U6 seems to be the only substrate of Mpn1, human Mpn1 also processes U6atac snRNA. PN cells bear unstable U6atac species with aberrantly long and oligoadenylated 3' ends. Our data corroborate the link between Mpn1 and snRNA stability suggesting that PN could derive from pre-mRNA splicing aberrations. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jul 2015 · FEBS letters
    0Comments 0Citations
    • "The diagnosis of our PN patients were made clinically and confirmed by molecular analysis for deleterious mutations in the causative gene, USB1, identified recently in 2010 [Volpi et al., 2010]. c.531delA homozygous deleterious mutation was detected in both patient's genomic DNA (Fig. 2C,D). "
    [Show abstract] [Hide abstract] ABSTRACT: Poikiloderma with neutropenia (PN), is a rare genodermatosis associated with patognomic features of poikiloderma and permanent neutropenia. Three common recurrent mutations of related gene, USB1, were considered to be associated with three different ethnic origins. The most common recurrent mutation, c.531delA, has been detected in seven Caucasian patients in the literature. In this paper, we present review of all patients from the literature and report two additional patients of Turkish ancestry with the diagnosis of PN. The diagnosis of these two PN patients were made clinically and confirmed by molecular analysis which detected the most common recurrent mutation, c.531delA. Genotype-ethnic origin correlation hypothesis, therefore, has been strengthened with this result. Short stature in PN, is a common finding, which until now has never been treated with growth hormone (GH). One of our patients is the first patient with attempted treatment of short stature via GH administration. Finally, both of our patients had high-pitched voice and vocal cord nodules which might be considered as additional clinical findings not associated with PN before. © 2014 Wiley Periodicals, Inc.
    Full-text · Article · Oct 2014 · American Journal of Medical Genetics Part A
    0Comments 2Citations
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