Clin Genet 2002: 61: 146151 Copyright C Munksgaard 2002Printed in Denmark. All rights reserved
RMRP gene sequence analysis confirms acartilage-hair hypoplasia variant with onlyskeletal manifestations and reveals a highdensity of single-nucleotide polymorphisms
Bonafe L, Schmitt K, Eich G, Giedion A and Superti-Furga A. RMRP L Bonafea, K Schmittb, G Eichc,gene sequence analysis confirms a cartilage-hair hypoplasia variant with A Giediona and A Superti-Furgaaonly skeletal manifestations and reveals a high density of single-nucleotide a Division of Metabolism and Molecularpolymorphisms. Paediatrics and Division of Radiology,Clin Genet 2002: 61: 146151. C Blackwell Munksgaard, 2002 University Childrens Hospital, Zurich,
Switzerland, b Landeskinderklinik Linz, Linz,Mutations in the RMRP gene that codes for an RNA subunit of the MRP Austria, c Division of Paediatric Radiology,RNAse complex are the cause of cartilage-hair hypoplasia (CHH; MIM Kantonsspital, Aarau, Switzerland250250). We tested the hypothesis that recessive metaphyseal dysplasiawithout hypotrichosis (M1M 250460), a disorder presenting with shortstature and metaphyseal dysplasia similar to CHH, but lacking hairanomalies, immunodeficiency and other extra skeletal features, might beallelic to CHH. We identified four mutation-carrying alleles segregatingwith the skeletal phenotype in two unrelated boys and their parents. Oneallele carried the common Finnish mutation pi70A G; the remainingthree carried pi195C T, pi238C T, and dupAAGCTGAGGACG at Key words: cartilage-hair hypoplasia 2. Sequencing 120 alleles from a control group revealed an unusually genetic mutations metaphysealhigh density of single-nucleotide polymorphisms in and around the dysplasia RMRP gene single-nucleotideRMRP gene: the biological significance of this finding is unclear. polymorphismsWe conclude that recessive metaphyseal dysplasia without hypotrichosis Corresponding author: Professor Andreais a variant of CHH, manifesting only as short stature and metaphyseal Superti-Furga, Division of Metabolism anddysplasia. Precise diagnosis of this form of metaphyseal dysplasia is not Molecular Paediatrics, University Childrenswithout importance because of recessive inheritance with corresponding Hospital, Steinwiesstrasse 75, CH-8032recurrence risk, as well as because of potential complications such as Zurich, Switzerland. Tel.: 41-1-266-7722;
fax: 41-1-266-7167;anaemia, susceptibility to infections and the increased likelihood ofe-mail: asuperti/access.unizh.chdeveloping cancer. The short stature and metaphyseal changes associated
with cone-shaped epiphyses of the hands should raise the diagnostic Received 29 October 2001, revised andpossibility of a CHH-related disorder that can then be confirmed by accepted for publication 3 Decembermutation analysis. 2001
The gene for cartilage-hair hypoplasia (CHH; met-aphyseal dysplasia, McKusick type; MIM 250250)has recently been identified (1). Alone out of allthe group of skeletal dysplasias, it codes not for aprotein, but for a RNA subunit of an RNAse com-plex. The pathogenesis of CHH is not yet under-stood, but RMRP may be a housekeeping gene,which would explain the pleiotropism of the dis-order.
A spectrum of mutations was identified in aseries of patients with classical CHH (1). Thesubjects were mostly of Finnish origin, but in-
cluded a Swiss child with severe CHH and im-munodeficiency who had undergone bone mar-row transplantation (2). There is a group of pa-tients who have short stature and radiographicchanges similar to those of CHH, but who alsohave normal hair, no immunodeficiency andnone of the other extra skeletal changes associ-ated with CHH [metaphyseal dysplasia withouthypotrichosis, MIM 250460 (3)]. The presentstudy was undertaken to test whether RMRPmutations could be identified in patients belong-ing to this group.
RMRP mutations in skeletal variant of CHH
Subjects and methodsSubjects
This boy was born at term to parents of Swiss andDanish extraction, and had a normal birth lengthof 52cm (P5090). The subjects growth declinedto P10 during the first year of his life and toslightly below P3 by 3years of age. At 7.5years ofage, he was 109cm in height, 8cm below P3 forSwiss children, but 2cm above P90 for Finnish pa-tients with CHH (4).
The subject was referred to us at 3years of agebecause of his short stature and moderate varusdeformity of the knees. Radiographic examinationat that time showed modest metaphyseal changes.Cartilage-hair hypoplasia was considered in thedifferential diagnosis and a haematological-im-munological work-up was performed. The sub-jects haemoglobin level was borderline low for hisage at 109gL1, and total IgG (4.23gL1) andtotal lgA (0.22gL1) in plasma were also border-line low. A more extensive work-up at 7years ofage showed a slightly decreased level of haemo-globin (110gL1), but normal values for severalparameters of cellular immunity (i.e. mitogen andantigen stimulations, lymphocyte subpopulations,expression of surface antigens, and adhesion mol-ecules). Therefore, an immune deficiency was ex-cluded.
The boys mother noted a susceptibility to highfever during childhood, but unusual or serious in-fections were never found. The subject had var-icella at 6years of age with pronounced efflor-escences, but no complication. Resection of hyper-trophic adenoids was done at 7years of age, alsowithout complications. He grows abundant hair ofdark blond to light brown colour that has neverbeen considered abnormal; hair thickness meas-urements have not been performed. Physical ex-amination showed moderately short stature withrelatively short legs (Fig.1) and mild joint laxity.The radiographic features are shown in Fig.1; al-though the diagnosis of a CHH variant was con-sidered at 3years of age, it was only at 7years thatmore characteristic features of CHH were seen,namely the metaphyseal irregularities and particu-larly the phalangeal cone-shaped epiphyses of thehand. The pelvis and the spine did not show pecu-liarities.
Case 2This boy was born at term to parents of Austrianorigin. His birth length was normal (48cm, P25),but his growth declined to slightly below P3 at 1(68.5cm) and 2years of age (77cm). At 3.2years of
age, the subjects height was 84cm (below P3 forthe reference paediatric population, but above P90for Finnish patients with CHH): his growth chan-nel has not changed since then, and at 10years ofage he was 116cm tall [13cm below P3 for con-trols, but 1cm above P90 for Finnish patients withCHH (4)].
The subject was referred to us for re-evaluationof his short stature (Fig.1). He had relatively shortlegs and laxity of the small joints of the hands.Radiographic examination revealed metaphysealstriations and phalangeal cone-shaped epiphysesof the hand (Fig.1). Metaphyseal dysplasias with-out hypotrichosis or a CHH variant were con-sidered in the differential diagnosis. A haematolog-ical-immunological work-up at 10years of ageshowed normal blood haemoglobin (126gL1),plasma IgG (9.25g l1), IgG subclasses and plasmalgA (1.03g l1) for his age. A lymphocyte subpopu-lation study gave normal results. There was no ab-normal susceptibility to infection during child-hood, with varicella infection producing severe ef-florescences but no complication, and the subjectsgeneral health has been excellent. As in case 1, hishair has always been considered normal; its thick-ness has not been measured.
Genomic DNA was obtained with informed con-sent from both patients and their parents, and ana-lysed by polymerase chain reaction (PCR) ampli-fication and direct sequencing of the RMRP gene.To obtain a reference sequence and ascertain poly-morphisms in the RMRP gene, genomic DNAfrom 60 anonymized unrelated controls of differ-ent ethnicities (27 Swiss, nine Italians, two French,five Germans, five Dutch, three British, one Span-ish, one Belgian, one Czech, one Austrian, fourAmericans and one Australian) was also studiedby PCR amplification and sequencing.
The sequence of the most 3 primers describedby M. Ridanp et al. (1) is not contained in thesequence deposited at GenBank (M29916). Thus,we amplified the RMRP gene using a new primerCHH-11-F, located 5 of the TATA box and the 3primer RM4R previously published by M. Rid-anp et al. (Fig.2). Polymerase chain reactionamplification of the RMRP gene as a single frag-ment of
Bonafe et al.
Fig.1. Clinical and radiographicfeatures of the two boys with a skeletalpresentation of cartilage-hairhypoplasia (CHH): Top row: Case 1 at 3years of age. There is moderate,proportionately short stature with mildvarus deformity of the legs. Note theabundant hair. Knee radiographs takenat 7years of age showing metaphysealirregularities with radiolucent areas andcoarser trabeculation at the knees. Handradiographs taken at the same ageshowing moderate metacarpalshortening, as well as multiplephalangeal epiphyses with half-moon ortrapezoid shapes (arrows) which are mostevident at the middle phalanges. Bottomrow: This photograph of case 2 (chosenby the patient himself) shows him at 9years of age. There is short stature, butno other features of CFF. Radiographsof the subjects knees at 10years of ageshowing metaphyseal changes which areslightly more pronounced than those seenin case 1, with striations extending fromthe metaphyses towards the diaphyses.The hand radiographs are similar tothose of case 1, but the changes areslightly more pronounced, possiblybecause of the subject is older. Themetacarpals are moderately shortenedand there are cone-shaped phalangealepiphyses with rounded half-moonshapes (arrows) throughout the proximaland medial phalangeal rows,occasionally with some irregularitiesproducing a trapezoid or tent-shapedappearance. The half-moon shapedcones are v