J Clin Immunol

DOI 10.1007/s10875-017-0385-7

ORIGINAL ARTICLE

Poikiloderma with Neutropenia in Morocco: a Report

of Four Cases
Ayoub Aglaguel 1 & Houria Abdelghaffar 1 & Fatima Ailal 2 & Norddine Habti 3 &
Sebastian Hesse 4 & Naschla Kohistani 4 & Christoph Klein 4 & Ahmed Aziz Bousfiha 2

Received: 9 November 2016 / Accepted: 16 March 2017

# Springer Science+Business Media New York 2017

Abstract homozygous mutations in the USB1 gene: c.609 + 1G>A in

Purpose Poikiloderma with Neutropenia (PN) is inherited
genodermatosis which results from a biallelic mutation in the
USB1 gene (U Six Biogenesis 1). PN, first described in Navajo
Native Americans, is characterized by early onset
poikiloderma, pachyonychia, palmo-plantar hyperkeratosis,
and permanent neutropenia. This condition results in frequent
respiratory tract infections during infancy and childhood. From
2011 to 2013, four cases of PN were diagnosed in Morocco. In
this paper, we report the first four cases of PN diagnosed in
Morocco, out of three unrelated consanguinous families.
Methods We investigated the genetic, immunological, and
clinical features of four Moroccan patients with PN from three
unrelated consanguinous families.
Results Mean age at onset was 3 months and mean age at
diagnosis was 7.5 years. The diagnosis of these PN patients
was made based on clinical features and confirmed by molec-
ular analysis for three cases. We identified two undescribed
* Ahmed Aziz Bousfiha
profbousfiha@gmail.com
1
Laboratory of Biosciences, Integrated and Molecular Functional
Exploration (LBEFIM), Faculty of Science and Techniques of
Mohammedia, Hassan II University of Casablanca,
Casablanca, Morocco
2
Clinical Immunology Unit, Department of Pediatrics, Abderrahim
Harouchi Children’s Hospital, CHU Ibn Rochd, LICIA Laboratory of
Clinical Immunology, Inflammation and Allergy, Faculty of
Medecine and Pharmacy, Hassan II University of Casablanca, Rue
Mohamed El Fidouzi, 20360 Casablanca, Morocco
3
Laboratory of Hematology, Cellular and Genetic Engineering,
Faculty of Medicine and Pharmacy, Hassan II University of
Casablanca, Casablanca, Morocco
4
Department of Pediatrics, Dr. Von Hauner Children’s Hospital,
Ludwig Maximilians University, Munich, Germany
two siblings and c.518 T>G(p.(Leu173Arg)) in the other case.
Conclusion This report confirms the clinical and genetic iden-
tity of Poikiloderma with Neutropenia syndrome.
Keywords Poikiloderma with Neutropenia . USB1 gene .
Moroccan . Mutation
Introduction
Poikiloderma is a chronic skin disorder that consists of telan-
giectatic lesions, areas of hypopigmentation and hyperpig-
mentation, and atrophy [1]. Poikiloderma occurs as the main
feature of several genodermatoses, the best known of which is
Rothmund–Thomson syndrome (RTS) [2]. In 1991, a new
syndrome of poikiloderma associated with neutropenia was
reported by Clericuzio et al. [3]. They reported 14 Navajo
Native Americans, including eight siblings from a large fam-
ily, developing in the first year of life of a popular erythema-
tous rash, which started on the limbs and spread over the trunk
and the face. This rash evolved into poikiloderma with a pro-
nounced acral involvement. All patients had recurrent bacte-
rial infections [3]. The first referred to as Navajo
poikiloderma; this syndrome is now known as Poikiloderma
with Neutropenia (PN, OMIM 604173) or Clericuzio-type
Poikiloderma with Neutropenia [4].
PN is a rare autosomal recessive genodermatosis, charac-
terized by early-onset poikiloderma, pachyonychia, palmo-
plantar hyperkeratosis, skeletal defects, and permanent neu-
tropenia [5]. The most prominent extracutaneous feature is an
increased susceptibility to infections, mainly affecting the re-
spiratory system, primarily due to a chronic neutropenia and
to neutrophil functional defects [3]. Since 2010, it has been
known that biallelic mutations in USB1 (U Six Biogenesis 1)
 J Clin Immunol

gene underlie Poikiloderma with Neutropenia [6]. This gene exons and flanking intron-exon boundaries of USB1
encodes U6 SnRNA Biogenesis Phosphodiesterase 1, an exo- gene (reference sequence ENST00000219281) were am-
nuclease active in processing spliceosomal U6snRNA [7, 8]. plified via the polymerase chain reaction (PCR) using
This protein is essential for the processing and stability of U6 the OneTaq Polymerase (New England Biolabs). The
snRNA, a molecule with a crucial role in RNA splicing [9]. primers and conditions used for PCR amplification are
U6 snRNA along with four other snRNAs (U1, U2, U4, and available upon request. Amplicons were checked by
U5) and their associated proteins make up the spliceosome electrophoresis in a 1% agarose gel and purified using
complex that catalyzes the removal of introns from mRNA. the QIAquick PCR Purification Kit (Qiagen) according
U6 is associated with the 5′ end of the intron by base pairing to the manufacturer’s protocol. PCR products were se-
before lariat formation [10]. Although USB1 functions as a quenced by dideoxynucleotide termination, with the
U6 biogenesis factor, the pathogenic mechanism of PN and BigDye Terminator v3.1 (Applied Biosystems) at
the role of USB1 in disease development remain to be fully GATC Biotech AG (Konstanz, Germany) with the same
explained. primers. Electrophoresis was performed onto the ABI
The varied symptoms of Poikiloderma with Neutropenia 3730xl genetic analyzer (Applied Biosystems). The raw
overlap with features of Rothmund–Thomson syndrome data were then analyzed with SeqMan Pro v10.1.1 soft-
(RTS) and Dyskeratosis Congenita (DC). DC is character- ware (Applied Biosystems) to be compared to the refer-
ized by the triad: nail dystrophy, hypo/hyper-pigmentation, ence sequence.
and oral leukoplakia caused by telomere defects [11].
Unlike PN, patients with DC have oral leukoplakia but
no persistent neutropenia. RTS is a rare autosomal reces-
sive disorder characterized by poikiloderma, congenital
skeletal abnormalities, short stature, premature aging, and
increased risk of malignant disease. RTS is caused by
mutations in the RECQL4 gene, which is believed to in-
terconnect with USB1 via SMAD4 proteins. This could
explain the partial clinical overlap between PN and RTS
[12]. Poikiloderma in RTS primarily occurs in sun-
exposed areas. In PN, however, initial localization of the
skin lesions includes extremities called acral presentation
[2]. The other distinctive feature is permanent neutropenia,
which is associated with PN.
To date, 50 patients have been reported in the literature
[13]. Our objective is to determine clinical manifestations,
immunological profile, and genetic defects for PN patients
diagnosed in Morocco. a b

Patients and Methods

Subjects

The study was conducted in accordance with the Helsinki

Declaration, with informed consent obtained from the pa-
tient’s family. Patients were recruited at the Clinical
Immunology Unit (CIU) in Casablanca and the Department
of Pediatrics of Rabat Children’s Hospital, between 2011 and
2013. The clinical diagnosis of PN was based on the presence
of poikiloderma and neutropenia.
c d
Mutation Analysis
Fig. 1 Panel showing some clinical features of the PN
syndrome poikiloderma (a), palmoplantar keratoderma (b) of
Genomic DNA was isolated from leukocytes using the patient 1, and palmoplantar keratoderma (c), atrophic scars (d)
standard phenol/chloroform method. All seven coding of patient 3
J Clin Immunol

Results
Subjects
Poikiloderma with Neutropenia, which combines dermatolog-
ical and immune disorders, belongs to the groups of congen-
ital neutropenia (CN), and it is therefore a primary immuno-
deficiency (PID). From 1998 to December 2014, a total of 502
patients with PIDs were registered, including 59 cases of CN.
In our series of CN, only four cases of PN (7% of CN) were
diagnosed in the last 2 years.
The four patients were born as term newborns, with ab-
sence of perinatal problems, and all growth parameters were
in the normal range. At birth, none of the presented patients
showed skin changes or dysmorphic signs.
infections in her first years of life. She had failure to thrive
since infancy, but no delay was observed in her psychomotor
development. Initial physical examinations revealed general-
ized poikiloderma on the trunk, extremities and face,
pachyonychia, failure to thrive, palmoplantar hyperkeratosis,
atrophic scars, sparse eyebrows, and dental caries (Fig. 1a, b).
She presented with hypogonadism and delayed puberty. Her
laboratory investigations showed neutropenia [(0.09–
0.83) × 109 L−1], lymphopenia [(1.04–1.82) × 109 L−1], de-
crease in NK cells, and polyclonal hypergammaglobulinemia.
Radiographic images revealed osteopenia. Computed tomog-
raphy (CT) of the chest showed bilateral bronchiectasis.
Patient 2

He was a 4.25-year-old boy. He came for the first time to the

Patient 1
She was an 11-year-old girl. The cutaneous manifestations
began at 1.5 months of age as a rash involving primarily the
extensor surface of lower extremities. We learned from the
medical history that she experienced recurrent pulmonary
Clinical Immunology Unit with his sister (patient 1). At
2 months, hypo- and hyper-pigmented skin lesions appeared
on both arms and slowly spread over the trunk and the face.
During the first years of life, he experienced recurrent pulmo-
nary infections. At the physical examination, he had
poikiloderma, palmoplantar hyperkeratosis, short stature,

Table 1 Clinical findings of PN patients

Family Family1 Family 2 Family 3

Patient Patient 1 Patient 2 Patient 3 Patient 4

Sex F M F F
Parental origin Morocco Morocco Morocco Morocco
Consanguinity 2nd degree 2nd degree 2nd degree 1st degree
Age at diagnosis 11 years 4.25 years 13 years 2.5 years
Poikiloderma + + + +
Onset 1.5 months 2 months 1 month 6 months
First localization Extremities Extremities Extremities Extremities
Persistent neutropenia + + + +
Recurrent pulmonary infections + + + +
Pachyonychia + − + −
Short stature + + + +
Palmoplantar keratoderma + + + +
Craniofacial dysmorphism − − − −
Osteopenia + + − −
Sexual development Delayed puberty with Normal Delayed puberty with Normal
hypogonadism hypogonadism
Hepatosplenomegaly − − − −
Myelodysplasia − − − −
Other findings Bilateral bronchiectasis, Dental caries, Bilateral Bronchiectasis, Lymphopenia
sparse eyebrows, Lymphopenia sparse eyebrows,
atrophic scars, dental atrophic scars, lymphopenia
caries, lymphopenia
Outcome Alive, age 14 years Alive, age 8 years Alive, age 15 years Alive, age 4.5 years

F Female M Male, Y Year, Mo Month

+ present, − absent
 J Clin Immunol

Lymphopenia (1.04–1.82) × 109 L−1

Neutropenia (0.31–1.38) × 109 L−1
normal mental and cognitive development, and dental caries.
His laboratory findings revealed neutropenia [(0.35–
1.24) × 109 L−1], lymphopenia [(1.54–2.95) × 109 L−1], and
polyclonal hypergammaglobulinemia. Radiographic images
revealed osteopenia.

Patient 3

Family 3

Patient 4
She was a 13-year-old girl. The skin manifestations appeared

ND
ND
at 1 month of age, starting from the face and the extensor

Lymphopenia (1.41–1.75) × 109 L−1

surface of the arms and then evolving into classical

Neutropenia (0.06–0.59) × 109 L−1

poikiloderma. The patient was referred to the Clinical
Immunology Unit due to recurrent pulmonary infections,
and skin lesions. Physical examination revealed generalized
poikiloderma, pachyonychia, palmoplantar hyperkeratosis,
atrophic scars, failure to thrive, and normal psychomotor de-
velopment (Fig. 1c, d). She had photosensitivity with sun

Low CD8
exposure. She presented with hypogonadism and delayed pu-

Family 2

Patient 3
berty. Laboratory investigations showed neutropenia [(0.06–

No
0.59) × 109 L−1], lymphopenia [(1.41–1.75) × 109 L−1], and
decreased number of CD8 cells. Chest CT showed bilateral

Lymphopenia (1.54–2.95/) × 109 L−1

Neutropenia (0.35–1.24) × 109 L−1
bronchiectasis.

Polyclonal [IgG, IgA = 2No]

Patient 4

She was a 2.5-year-old girl. At 6 months, parents noticed

papular erythematous rash started on limbs which spread over
the trunk later and lastly the face. She had the history of re- Patient 2
current infections, particularly pulmonary infections. On the
physical examination, she had short stature, poikiloderma, and

No
palmoplantar hyperkeratosis. Neutropenia [(0.31–
1.38) × 109 L−1] and lymphopenia were found [(1.04–
Lymphopenia (1.04–1.82) × 109 L−1
Neutropenia (0.09–0.83) × 109 L−1

1.82) × 109 L−1] at the time of presentation.

Polyclonal [IgG, IgA = 2No]

The clinical and laboratory findings in these PN patients

are listed in Tables 1 and 2, respectively.

Mutation Analysis
Low CD19

Molecular analysis was available for two families (three pa-

Patient 1
Family1

tients). Patient 4 has not been processed for molecular analy-

Summary of laboratory investigations

sis. Mutations were found in both alleles for all patients and
are summarized in Table 3. We found two novel mutations.
(CD3+,CD4+,CD8+,CD19+,CD16/56+)

Discussion
No Normal, ND not done
Immunoglobulin profiles

A clinical diagnosis of PN was made in four patients. In three

of these patients, the diagnosis was established by molecular
Lymphocyte subsets
Blood cell counts

analysis of the USB1 gene. Mutation analysis revealed two

undescribed homozygous mutations in the USB1 gene. In pa-
tients 1 and 2, we identified homozygosity for a donor splice
Table 2

Family

Patient

site mutation (c.609 + 1G>A), predicted to cause a frameshift

and premature termination. The unaffected parents were
J Clin Immunol

Table 3 USB1 mutations in PN patients

Location Codon Change type Protein change Patient (family)

Intron 5 c.609 + 1G>A Splice site (donor) Presumable frameshift and Patients 1 and 2 (family 1)
premature protein truncation
Exon 5 c.518 T>G Missense p.(Leu173Arg): Protein function Patient 3 (family 2)
might get lost due to an altered splice site

heterozygous for the mutation. The biallelic missense muta-
tion c.518 T>G(p.(Leu173Arg)) was found in patient 3. The
parents carry the same mutation in heterozygous state. This
T>G substitution at the 15th nucleotide of exon 5 affects the
Leu173 residue of the GLEV domain. This domain is highly
conserved among vertebrates. The Human Splicing Finder
(HSF 3.0) predicts a potential alteration of splicing (activation
of an exonic cryptic donor site adding leading to loss of 92
nucleotides of exon 5 and frameshift). MutationTaster predicts
that His208 in the active site and the modified residue N6-
acetyl-Lys 258 might get lost (due to missplicing and frame-
shift) leading to loss of protein function.
Interestingly, several other PN patients originating from
Morocco have previously been reported [14]. In 2012,
Colombo et al. reported an Algerian patient with c.179delC
homozygous mutation in exon 2 [15]. This deletion has been
already described in three Moroccan siblings, suggesting ei-
ther common ancestry or a founder effect [16]. Table 4 sum-
marizes the clinical findings of PN North-West African pa-
tients. Like all PN patients described in literature, Moroccan
patients had poikiloderma and neutropenia as the main fea-
tures of the syndrome.
Concerning the treatment of PN, there is little information
on the use of Granulocyte-Colony Stimulating Factor (G-
CSF) for neutropenia. A good response to G-CSF has been
reported in only two patients [4, 15]. Growth Hormone (GH)
therapy for the treatment of short stature was applied to a PN
patient and no significant response was observed [17]. Further
practices are needed to demonstrate the effects of the GH
therapy.
Like all PID, PN is underdiagnosed in the world, and es-
pecially in low-income countries. Among 512 cases of PID
registered in Morocco since 1988, only four patients were
diagnosed with PN [18]. The high rate of consanguinity in
Morocco, estimated at 15.25% in general population, suggests
high incidence of autosomal recessive genetic disorders [19].
We speculate that the true number of Moroccan patients with
PN may be much higher, and that a lack of awareness ob-
served in our medical community, the lack of diagnostic facil-
ities in certain regions, delay in diagnosis, and difficulties to
access modern health care may lead to underdiagnosis and
even early death of affected patients.
In summary, PN is a rare autosomal recessive
genodermatosis associated with susceptibility to infections
and mutations in the USB1 gene. All our patients showed
the typical clinical features of PN. We also showed a large
delay in diagnosis in our series. Further studies on large cohort
are needed to determine the true incidence and prevalence of

Table 4 Summary of clinical and molecular findings in PN North-West African patients

Reference Tanaka et al. [16] / Mostefai et al. [14] Our patients Colombo et al. [15]

Origin Morocco Algeria

Mutation c.179delC c.609 + 1G>A c.518 T>G ND c.179delC

Clinical features Poikiloderma + + + + + + + +

Neutropenia + + + + + + + +
Recurrent infections + + + + + + + +
Nail disorders + + − + + + − +
Short stature + + − + + + + −
Craniofacial dysmorphism − − − − − − − −
Palmoplantar hyperkeratosis + + − + + + + +
Dental defects + + + + + − − −
Anemia − − − + − − + −

ND not done
+ present, − absent

the disease and find out whether there are more common mu-
tations among Moroccan PN patients.
Acknowledgements The authors would particularly like to thank the
patients and their families, whose trust, support, and cooperation were
essential for the collection of the data used in this study. The authors
would also like to thank the HAJAR association (http://www.hajar-
maroc.org) and the Moroccan Society for Primary Immunodeficiencies
(http://www.pid-moroccansociety.org) for their helpful support.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
Informed Consent All procedures followed were in accordance with
the ethical standards of the responsible committee on human experimen-
tation (institutional and national) and with the Helsinki Declaration of
1975, as revised in 2000 (5). Informed consent was obtained from all
patients included in the study.
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