Poikiloderma with Neutropenia in Morocco: a Report of Four Cases

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ORIGINAL ARTICLE

Poikiloderma with Neutropenia in Morocco: a Report of Four Cases

Ayoub Aglaguel¹&Houria Abdelghaffar¹&Fatima Ailal²&Norddine Habti³&

Sebastian Hesse⁴_&Naschla Kohistani⁴_&Christoph Klein⁴_&Ahmed Aziz Bousfiha²

Received: 9 November 2016 / Accepted: 16 March 2017

#Springer Science+Business Media New York 2017

Abstract

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

homozygous mutations in the

USB1

gene: c.609 + 1G>A in 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)

* 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

DOI 10.1007/s10875-017-0385-7

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gene underlie Poikiloderma with Neutropenia [6]. This gene encodes U6 SnRNA Biogenesis Phosphodiesterase 1, an exo- nuclease active in processing spliceosomal U6snRNA [7,

8].

This protein is essential for the processing and stability of U6 snRNA, a molecule with a crucial role in RNA splicing [9].

U6 snRNA along with four other snRNAs (U1, U2, U4, and U5) and their associated proteins make up the spliceosome complex that catalyzes the removal of introns from mRNA.

U6 is associated with the 5′ end of the intron by base pairing before lariat formation [10]. Although USB1 functions as a U6 biogenesis factor, the pathogenic mechanism of PN and the role of USB1 in disease development remain to be fully explained.

The varied symptoms of Poikiloderma with Neutropenia overlap with features of Rothmund–Thomson syndrome (RTS) and Dyskeratosis Congenita (DC). DC is character- ized by the triad: nail dystrophy, hypo/hyper-pigmentation, 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.

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.

Mutation Analysis

Genomic DNA was isolated from leukocytes using the

exons and flanking intron-exon boundaries of

USB1

gene (reference sequence ENST00000219281) were am- plified via the polymerase chain reaction (PCR) using the OneTaq Polymerase (New England Biolabs). The primers and conditions used for PCR amplification are available upon request. Amplicons were checked by electrophoresis in a 1% agarose gel and purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s protocol. PCR products were se- quenced by dideoxynucleotide termination, with the BigDye Terminator v3.1 (Applied Biosystems) at GATC Biotech AG (Konstanz, Germany) with the same primers. Electrophoresis was performed onto the ABI 3730xl genetic analyzer (Applied Biosystems). The raw data were then analyzed with SeqMan Pro v10.1.1 soft- ware (Applied Biosystems) to be compared to the refer- ence sequence.

a b

c d

Fig. 1 Panel showing some c linical features of the PN syndrome poikiloderma (a), palmoplantar keratoderma (b) of patient 1, and palmoplantar keratoderma (c), atrophic scars (d)

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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.

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

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) × 10

⁹

L

⁻¹

], lymphopenia [(1.04–1.82) × 10

⁹

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 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 hypogonadism

Normal Delayed puberty with

hypogonadism

Normal

Hepatosplenomegaly − − − −

Myelodysplasia − − − −

Other findings Bilateral bronchiectasis, sparse eyebrows, atrophic scars, dental caries, lymphopenia

Dental caries, Lymphopenia

Bilateral Bronchiectasis, sparse eyebrows,

atrophic scars, lymphopenia

Lymphopenia

Outcome Alive, age 14 years Alive, age 8 years Alive, age 15 years Alive, age 4.5 years

FFemaleMMale,YYear,MoMonth +present,−absent

1.38) × 10

⁹

L

⁻¹

] and lymphopenia were found [(1.04–

1.82) × 10

⁹

L

⁻¹

] at the time of presentation.

The clinical and laboratory findings in these PN patients are listed in Tables

1

and

2, respectively.

Mutation Analysis

Molecular analysis was available for two families (three pa- tients). Patient 4 has not been processed for molecular analy- sis. Mutations were found in both alleles for all patients and are summarized in Table

3. We found two novel mutations.

Discussion

A clinical diagnosis of PN was made in four patients. In three of these patients, the diagnosis was established by molecular 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

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

_b^le

2Summaryoflaboratoryinvestigations milyFamily1Family2Family3 tientPatient1Patient2Patient3Patient4 dcellcountsNeutropenia(0.09–0.83)×109L−1 Lymphopenia(1.04–1.82)×109L−1Neutropenia(0.35–1.24)×109L−1 Lymphopenia(1.54–2.95/)×109L−1Neutropenia(0.06–0.59)×109L−1 Lymphopenia(1.41–1.75)×109L−1Neutropenia(0.31–1.38)×109L−1 Lymphopenia(1.04–1.82)×109L−1 profilesPolyclonal[IgG,IgA=2No]Polyclonal[IgG,IgA=2No]NoND ymphocytesubsets (CD3+,CD4+,CD8+,CD19+,CD16/56+)LowCD19NoLowCD8ND Normal,NDnotdone

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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.

I n s um m a r y, P N i s a ra r e a ut oso m a l r ec es s i v e 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 3 USB1mutations in PN patients

Location Codon Change type Protein change Patient (family)

Intron 5 c.609 + 1G>A Splice site (donor) Presumable frameshift and premature protein truncation

Patients 1 and 2 (family 1)

Exon 5 c.518 T>G Missense p.(Leu173Arg): Protein function

might get lost due to an altered splice site

Patient 3 (family 2)

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 − − − + − − + −

NDnot done +present,−absent

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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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