Atypical Rothmund-Thomson syndrome in a patient with compound ...

Eur J Pediatr (2008) 167:175–181 DOI 10.1007/s00431-007-0447-6

ORIGINAL PAPER

Atypical Rothmund-Thomson syndrome in a patient with compound Heterozygous Mutations in RECQL4 Gene and phenotypic features in RECQL4 syndromes Yves Sznajer & H. Annika Siitonen & Gaia Roversi & Chantal Dangoisse & Michèle Scaillon & France Ziereisen & Sylvie Tenoutasse & Marjo Kestilä & Lidia Larizza

Received: 21 November 2006 / Revised: 13 February 2007 / Accepted: 14 February 2007 / Published online: 20 March 2007 # Springer-Verlag 2007

Abstract We describe the natural history of the RTSII phenotype in a 7-year-old boy who developed intrauterine and postnatal growth retardation, failure to thrive and persisting diarrhoea. The growth hormone stimulation test identified an isolated growth hormone deficiency. Since infancy, the patient manifested skin lesions characterized by a very mild poikilodermic-like appearance on the cheeks only, widespread café-au-lait spots and the absence of eyebrows and eyelashes. There was no cataract. Orthopaedic and radiologic work-up identified the absence of thumb anomaly and radial head luxation and patellar hypoplasia. Neurologic, cognitive milestones and intelligence were normal. The cytogenetic work-up did not show any anomaly. Based on this clinical presentation, we carried out a sequencing analysis

of the RECQL4 gene, which is responsible for RothmundThomson, RAPADILINO and Baller-Gerold syndromes and found a splice site mutation (IVS10-1G>A) and a nucleotide substitution in exon 12 (L638P). The mother was identified as a carrier for the substitution in exon 12 and the father for the splice site mutation, respectively. An analysis of the transcripts focused on the RECQL4 helicase domain: in the proband only those generated from the maternal L638 allele were present. This case report emphasizes the clinical overlap between RAPADILINO and Rothmund-Thomson syndromes within a continuum phenotypic spectrum. The distinctive set of clinical signs displayed by the patient may be accounted for by his unique combination of two different RECQL4 mutations. The molecular findings provide information that

Y. Sznajer (*) Clinical Genetics, Hôpital Universitaire des Enfants Reine Fabiola, Unité de Génétique Clinique and Center for Human Genetics, Université Libre de Bruxelles, 15 avenue J.J Crocq, 1020 Brussels, Belgium e-mail: [email protected]

M. Scaillon Gastroenterology, Hôpital Universitaire des Enfants Reine Fabiola, Université Libre de Bruxelles, Brussels, Belgium

H. A. Siitonen : M. Kestilä Department of Molecular Biology, National Public Health Institute, Helsinki, Finland G. Roversi : L. Larizza Division of Medical Genetics, San Paolo School of Medicine, University of Milan, Milan, Italy C. Dangoisse Dermatology, Hôpital Universitaire des Enfants Reine Fabiola, Université Libre de Bruxelles, Brussels, Belgium

F. Ziereisen Radiology, Hôpital Universitaire des Enfants Reine Fabiola, Université Libre de Bruxelles, Brussels, Belgium S. Tenoutasse Endocrinology, Hôpital Universitaire des Enfants Reine Fabiola, Université Libre de Bruxelles, Brussels, Belgium

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enhances our comprehension of genotype-phenotype correlations in RECQL4 diseases, enables a more precise genetic counseling to the parents and facilitates a more appropriate long-term follow-up to the affected child. Keywords RAPADILINO . RECQL4 mutations . Rothmund-Thomson syndrome

Introduction When the original descriptions by Rothmund [21] and Thomson [24] were combined into Rothmund-Thomson syndrome (RTS; MIM 268400) by Taylor, ‘poikiloderma congenitale’ remained as the only required symptom for the diagnosis [23]. Since then, it has emerged that RTS patients represent a phenotypically and genotypically heterogeneous group of patients [11, 32]. The natural history of the socalled RTSII phenotype is characterized by poikiloderma associated with additional symptoms of variable severity over time: growth retardation, gastrointestinal problems, failure to thrive, radial ray defects, osteosarcoma, alopecia and the loss of eyebrows and eyelashes. Most of the RTSII patients carry RECQL4 gene mutations [10, 32], but mutations in the RECQL4 gene are also responsible for the RAPADILINO (MIM 266280) [22] and Baller-Gerold syndromes (BGS; MIM 218600) [27]. These three ‘RECQL4 syndromes’ share as overlapping features intrauterine and postnatal growth retardation and bone malformations, such as hypoplasia and/or aplasia of the thumbs, radius and patellae [4, 15, 26]. Poikiloderma, the hallmark feature of RTS, is also found in BGS patients but not in cases of RAPADILINO. The differential diagnosis between RTS and BGS relies on the presence of craniosynostosis in the latter. The role of RECQL4 protein is largely unknown [19], and unlike other human RECQ homologues, RECQL4 does not possess helicase activity [14]. However, RECQL4 has been postulated to be implicated in the maintenance of genomic stability, which could explain the increased susceptibility of patients with the ‘RECQL4 syndromes’ to osteosarcoma, especially RTSII patients [19]. We report here on a propositus who developed cardinal features associated with the spectrum of ‘RECQL4 syndromes’. Although the patient presented with mild limb anomaly and slight skin involvement, the overall clinical picture prompted RECQL4 gene mutation screening.

Case report The propositus is a young boy, the first child of healthy unrelated Caucasian parents. Intrauterine growth retardation

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of an unidentified aetiology was recorded from the second trimester of pregnancy. Following a full term pregnancy, delivery was normal. At birth, the weight, height and head occipitofrontal circumference (OFC) of the infant were 2025 g (−4.2 SD); 45 cm (−3.3 SD) and 31.6 cm (−2.6 SD), respectively. Chronic diarrhoea and feeding difficulties were noted from infancy. Non-bloody stools six to eight times a day persisted throughout childhood. For 5 years, gavage feeding by means of gastrostomy during the nights was the only option that would enable the child to meet his appropriate caloric intake. Repeated digestive tract endoscopies did not identify any mucosal inflammation, infection or any proliferative disease. There was no evidence of a malabsorptive process. Esophageal, gastric, intestinal and colic biopsies did not reveal any histologic anomaly. Psychomotor milestones, intelligence and behavior were normal. There was neither malformation of the limbs nor anomalies of the thumb anomaly (Fig. 1a), but bilateral radial head luxation (Fig. 1b) was identified on orthopaedic and radiologic work-up. Rotation movements of the forearms were thus limited. The patellae were palpable from birth but not visible on X-rays until the age of 7 years, at which time they appeared to be hypoplastic (Fig. 1c). A poikiloderma-like appearance of the skin of his cheeks was present only since the age of 5 months. His hair was sparse, thin and brittle, while his eyebrows and eyelashes were absent. Café au lait spots were present on the upper part of the trunk from the age of 3 years onwards. There was no skin sensitivity to sun exposure; lymphedema on the dorsal part of his hands and feet was present from birth until the age of 6 years. The oral cavity presented a high arched palate, and first teeth eruption was delayed (15 months). The ophthalmologic anterior chamber and fundus were normal. (Fig. 2a–e) There was no history of infections, and screening for cellular or humoral immune deficiency remained negative. Due to a failure to thrive, an endocrine evaluation was carried out, which identified an isolated growth hormone deficiency; growth hormone replacement therapy was then commenced. Cerebral computed tomography (CT) scans and magnetic resonance images (MRI) were normal. The standard karyotype was 46,XY, and screening for chromosome breakage anomaly (Mitomycin C) was negative. At the time of writing this article, the child was 7 years old. He has a normal intelligence but has not developed cataract, has persistent diarrhoea (twice a day), a short stature (height is −3.5 SD) and upper limb limited rotation due to bilateral radial head luxation. Our differential diagnostic procedure focused on RAPADILINO or RTS. Analysis of the RECQL4 gene identified one splice site mutation (IVS10-1G>A) and one nucleotide transition g.3796T>C that caused an amino acid substitution, L638P, in exon 12. The mother was identified as a carrier for the nucleotide substitution and the father for the splice site

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panel), electrophoresis of the RT-PCR products from exons 5–13 encoding the RECQL4 portion, which includes the helicase domain, showed a band of the expected size (484 bp) in both the patient and the control. A band pattern that was identical for both the patient and the control was also obtained by amplifying the region of interest with two additional different primer pairs (data not shown) [2]. To check whether the observed standard-size RT-PCR product might be attributed to both mutant alleles or only to that carrying the missense mutation g.3796T>C (L638P), we excised the 484-bp bands of the patient and control from the gel and sequenced them. As shown in Fig. 3 (right panel), the presence of the expected T allele has been completely replaced in the proband by transcripts originating from the maternal mutant C allele, thereby demonstrating that there are no correctly spliced transcripts derived from the paternally inherited IVS10-1G>A allele.

Methods

Fig. 1 a Hand radiograph of the patient at the age of 7 years and 4 months, showing the absence of distal radio-cubital anomaly; normal first ray. Bone age is normal for his age based on Greulich and Pyle tables. b The forearm of the patient, showing proximal radial head luxation. c Lower limb radiographs revealing possible patellar hypoplasia at the age of 7 years and 4 months

mutation. In order to detect possible splicing aberrations related to the identified splice site mutation (IVS10-1G>A), we performed an analysis of the transcripts by reverse transcriptase (RT)-PCR on fibroblasts from the patient setup culture. The destruction of the IVS 10 splice acceptor may have different outcomes, including IVS 10 retention (+79 bp), exon 11 skipping (−174 bp) or the activation of alternative cryptic splice sites. As can be seen in Fig. 3 (left

The complete RECQL4 gene (exons, exon–intron boundaries and all introns with the exception of intron 12) was amplified by PCR and fully sequenced according to Siitonen et al. [22]. Both mutations were found in the same PCR fragment, which we were able to obtain using primers ex10-12-F (5′-GCAGCAGATCTGGGATGACT-3′) and ex10-12-R (5′- GCAGTGGGGAGTGAGGAG-3′). Total RNA was extracted from fibroblast cultures obtained from the skin biopsy of the patient and from a normal control according to the manufacturer’s instructions (TRI-Reagent, Sigma, St. Louis, Mo.). cDNA synthesis was performed using gene-specific primer RECQL-4R (5′-ACTGCCCTAGCCTCTGACAA-3′) for 1 h at 59°C, followed by PCR with primers RECQL42Fa (5′-TCTCTCCCCTGCTGTCACTC-3′) (exon 5) and RECQL4-2R (5′-AGCGTCAACAGTGCCTGGTC-3′) (splice site junction exons 12–13) at the following conditions: one cycle at 94°C for 4 min; 35 cycles at 94°C for 30 s; 60°C for 25 s; 72°C for 20 s; a final extension of 2 min at 72°C (ThermoScript RT-PCR System with platinum Taq DNA polymerase; Invitrogen, Carlsbad, Calif.). After excision and purification from agarose gel (QIAquick Gel Extraction kit; QIAGEN, Valencia, Calif.), the PCR products were directly sequenced using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, Calif.) on a capillary sequencer (Spectrumedix, State College, Pa.). Protein amino acid sequences were aligned by using the CLUSTALW 1.8 alignment program (http:// searchlauncher.bcm.tmc.edu/multi-align/multi-align.html). RECQL4 amino acid sequences were collected from the

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Fig. 2 a General facial and upper trunk appearance of the propositus at age 5 years: hair is sparse and thin; eyebrows and eyelashes are lacking; long nose and telangiectatic-like skin lesions are present on cheeks (poikiloderma). Status post-gastrostomy is visible on left abdominal region. b Upper right arm focusing on brownish spots. c Lateral view at the same age underlines the sparse, slowgrowing hair and milder caféau-lait spots around the external ear. Poikiloderma on cheeks appears to be very mild. d Face of the propositus at age 6 years: poikiloderma on cheeks seems to have disappeared; hair is unchanged; eyebrows and eyelashes are missing; there is no cataract. e General upright appearance: note the resting position of the upper forearms (due to radial head luxation) as well as feet abduction resulting from tibial torsion. Post-gastrostomy status can be seen. Poikiloderma is not noted on any other body part

NCBI database (http://www.ncbi.nlm.nih.gov/), with the following accession numbers: BAA86899.1 Homo sapiens, NP_478121.1 Mus musculus, XP_216973.3 Rattus norvegicus, CAD58810.1 Bos taurus, AAY89585.1 Xenopus laevis and NP_652607.1 Drosophila melanogaster.

Fig. 3 Left panel Electrophoresis of the reverse transcriptase (RT)-PCR products encompassing exons 5–13; note the band of the expected size (484 bp) in both the patient and normal control. Right panel Electropherograms showing the normal homozygous condition (T/T) in the control (bottom) and the loss of the transcripts from the normal allele, with only those from the mutant allele (−/C) present in the patient (top)

Discussion The patient showed some of the symptoms of ‘RECQL4 syndromes’, even though the radial ray defects were subclinical and the distribution of poikiloderma was atypical.

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The differential diagnosis ruled out BGS based on the absence of craniosynostosis, while RAPADILINO and RTSII were both assessed as being reasonable diagnoses. The RAPADILINO syndrome was suggested, which is an eponym based on the occurrence of specific features in three sporadic cases and a pair of sibs, namely: RAdial aplasia/hypoplasia-high PAlate-PAtellar aplasia-DIarrhoeaLIttle size-LImb anomaly-NOrmal intelligence-long slender NOse [6]. Symptoms such as radial ray defects and short stature are also found in RTSII and BGS patients and represent the most common features found in the ‘RECQL4 syndromes’ [11]. Only a few sporadic cases of RAPADILINO syndrome have been reported to date [4, 5, 7, 8, 20, 22]; of these, the same phenotypic variation was apparent. One patient initially diagnosed with RAPADILINO syndrome was later re-diagnosed with RTS. [4, 7] The only dermatological symptoms found in RAPADILINO patients are the irregular-shaped brownish spots [5, 6], and it is likely that mildly affected RAPADILINO patients have either not been diagnosed or not reported. Most of the RAPADILINO patients have characteristic refractory diarrhoea that persists for years, but the natural history and precise description of the syndrome are lacking [6, 7, 22, 28]. Vargas et al. described a boy who developed cardinal features of RAPADILINO but for whom a postnatal growth retardation was attributed to malabsorptive diarrhoea [28]. This symptom has also been described among RTSII and BGS patients, but to our knowledge, only three RTSII patients have had as severe gastrointestinal problems as the patient described herein [4]. It has been postulated that digestive tract involvement possibly emerges from an autoimmune process since several auto-antibodies (antinu-

clear, smooth muscle, thyreoglobulin, enterocytes) have been present in one sporadic case [4]. In the present case, in which the patient has only very mild upper limb involvement, if we had followed the Kääriäinen work-up on radial ray defect we would have directed the diagnosis to RAPADILINO syndrome [6] (and then to the molecular screening for RECQL4 gene mutation). What makes RTS distinctive from RAPADILINO is the poikilodermatous rash, alopecia and absence of eyelashes and eyebrows. The following findings have been reported on 200 RTS patients mined from the literature: of the 143 reports specifying age of onset, skin changes were noted during the first year of life in 89%, limb anomaly was associated in two-thirds of the patients and cataract in 30% (61/200), whereas onset occurred before age 6 years (52/ 61). Isolated growth hormone deficiency is an unusual finding, whereas gastroenteritis with digestive tract involvement was not mentioned in the extensive review by Vennos [29]. More recently, Wang et al. delineated clinical findings in 41 patients with RTS: poikilodermatous rash was present in all (onset: 3–10 months), 50% had sparse scalp hair and 73% had sparse eyebrows and eyelashes; 75% developed skeletal anomalies (osteopenia, fractures, dislocations, irregular metaphyses, stippled patellae ossification and radial ray defect: thumb appendage, bifid thumb, missing thumb). More intriguingly, only two of 32 patients developed ophthalmologic anomaly (i.e. cataract). Growth failure was present in 66% (25/38), and a follow-up screening program detected sarcoma in 32% (13/41) [30]. Among the RTSII, RAPADILINO and BGS patients, 31/36 (86%) had skeletal anomalies; 32/34 (94%) manifested growth failure; 10/32 (31%) had alopecia, sparse hair and/

Table 1 Clinical featuresa associated with the Baller-Gerold, Rothmund-Thomson, RAPADILINO syndromes and with the present patient Clinical feature

Baller-Gerold syndrome

Rothmund-Thomson syndrome

RAPADILINO syndrome

Present case

Short stature Coronal and/or lambdoid craniosynostosis Sudden infant death Anal anomaly Malformed ears Poïkiloderma Sparse hair Sparse eyebrow/eyelashes Cataract Diarrhoea/feeding problems Normal intelligence Patellar hypoplasia/aplasia Radial ray Ddfect Thumb anomaly Osteosarcoma

Almost constant Constant 25% in First year of life Anteposition, Posteriorly Rotated, Unusualb Absent Absent Absent Absent 50% Mental retardation Absent Bilaterally symmetric Constant Absent

66% Absent Absent Unconstant Absent Constant 50% 75% 60% A, leading to missplicing, and g.3796T>C, leading to amino acid substitution L638P. Proline is a ring-structured amino acid and the change from leucine could lead to a conformational change in the way the protein is folded. In addition, this mutation occurs in the so-called region III of the helicase domain [9] and affects an amino acid residue highly conserved in the human, mouse, rat, cow, African clawed toad and fruit fly genomes. No precise correlation could be made between the unusual combination of the above splicing and missense mutations and the atypical poikiloderma [2]. Mutations leading to missplicing as well as IVS10-1G>A (g3427G>A; g3865G>A; g3712del24) have been reported in RTS patients and are considered to be truncating mutations

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[32]. RT-PCR analyses did not reveal the presence of aberrant transcripts attributable to missplicing. To the contrary, the sequencing analyses of the expected-sized RT-PCR products revealed only the presence of correctly spliced RNA products that could be attributed to the presence of the maternal allele carrying the g.3796T>C (L638P) mutation. Based on these analyses, we conclude that whatever the effects of the IVS10-1G>A mutation on missplicing, the paternal IVS10-1G>A transcripts are probably subjected to nonsense-mediated mRNA decay, leading to a haplo-insufficiency for the correctly spliced maternal mRNA. The role of the RECQL4 deficient human cells is currently being unraveled, but only gross parameters have yet been defined in terms of classifying the effects of different RECQL4 mutations [17, 18]. Another level of complexity emerges from the unique combination of different mutated alleles in compound heterozygous patients. Cases such as these described here provide useful information that enable the clinician to address the genotype-phenotype correlation in ‘RECQL4 syndromes’ patients. In particular, long-term follow-up is now provided for the increased risk of osteosarcoma in the presence of at least one RECQL4 truncating mutation.

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