European Journal of Human Genetics (2008) 16, 897–905 & 2008 Nature Publishing Group All rights reserved 1018-4813/08 $30.00 www.nature.com/ejhg ARTICLE Molecular and cytogenetic analysis of the spreading of X inactivation in a girl with microcephaly, mild dysmorphic features and t(X;5)(q22.1;q31.1) Roberto Giorda*,1,7, M Clara Bonaglia2,7, Greta Milani1, Anna Baroncini3, Francesca Spada3, Silvana Beri1, Giorgia Menozzi4, Marianna Rusconi1 and Orsetta Zuffardi5,6 1Molecular Biology Laboratory, ‘E. Medea’ Scientific Institute, Bosisio Parini, Lecco, Italy; 2Cytogenetics Laboratory, ‘E. Medea’ Scientific Institute, Bosisio Parini, Lecco, Italy; 3UO Genetica Medica, AUSL Imola, Imola, Italy; 4Bioinformatic Laboratory, ‘E. Medea’ Scientific Institute, Bosisio Parini, Lecco, Italy; 5Biologia Generale e Genetica Medica, Universita` di Pavia, Pavia, Italy; 6IRCCS Policlinico San Matteo, Pavia, Italy X chromosome inactivation involves initiation, propagation, and maintenance of gene inactivation. Studies of replication pattern and timing in X;autosome translocations have suggested that X inactivation may spread to autosomal DNA. To examine this phenomenon at the molecular level, we have tested the transcriptional activity of a number of chromosome 5 loci in a female subject with microcephaly, mild dysmorphic features and 46,X,der(X)t(X;5)(q22.1;q31.1) karyotype. RT-PCR analysis of 20 transcribed sequences spanning 5q31.1-qter revealed that nine of them were not expressed in somatic cell hybrid clones carrying the translocated chromosome. However, eight genes were expressed and therefore escaped inactivation. This direct expression test demonstrates that spreading of inactivation from the X chromosome to the adjoining autosomal DNA was incomplete and ‘patchy’. Inactivation was associated in most instances to methylation of the CpG sequences in genes containing CpG islands, but was also present in CpG islandless genes. These results agree with those obtained for other X;autosome translocations and demonstrate that autosomes are partially resistant to Xist-mediated spreading and/or maintenance of inactivation. Repeat distribution analysis does not suggest an association between L1 and LINE repeat density on chromosome 5 and gene inactivation. The expression data may also explain why the proband manifests an attenuated clinical phenotype compared to subjects with partial chromosome 5 trisomy. European Journal of Human Genetics (2008) 16, 897–905; doi:10.1038/ejhg.2008.28; published online 27 February 2008 Keywords: unbalanced translocation; chromosome X inactivation; gene expression; spreading of inactivation Introduction sexes. It randomly silences the majority of genes on one X X-chromosome inactivation is a mechanism of compensa- chromosome in somatic cells of female mammals. The tion for the gene dosage differences that exist between the inactive status is stable and maintained through subse- quent cell divisions. The mechanism by which X inactiva- tion occurs is still not entirely clear. In mammals, it is regulated by a single cis-acting locus, the X inactivation *Correspondence: Dr R Giorda, Molecular Biology Laboratory, ‘E. Medea’ Scientific Institute, via don Monza 20, Bosisio Parini, Lecco 23842, Italy. centre (Xic). The Xic transcribes a non-coding RNA, the X Tel: þ 39 031 877911; Fax: þ 39 031 877499; inactive-specific transcript (Xist), which associates with E-mail: [email protected] 7 the chromosome in cis and converts the chromosome to These first two authors should be regarded as joint first authors. 1–5 Received 4 October 2007; revised 11 January 2008; accepted 17 January a silent heterochromatic configuration. Epigenetic 2008; published online 27 February 2008 changes associated with inactive X chromosomes include Spreading of X inactivation in a t(X;5) R Giorda et al 898 hypermethylation of CpG islands, covalent modification and impaired fine motor skills. From the time of starting of core histone tails, and incorporation of variant school she had learning difficulties and low tolerance histones.6 frustration. When the Xic is translocated onto autosomes, spreading On examination at age 7 1/2 years, she was 130 cm tall of the silent chromatin structure into autosomal chroma- (90th centile), weighed 36 Kg (497th centile) with a head tin can occur,7 but is frequently attenuated and incom- circumference of 48 cm (o3rd centile). She had brachyce- plete.8–10 Initial observations in X;autosome unbalanced phaly (cephalic index ¼ 85.2%), upslanting palpebral translocations have noted the inactivation of single fissures, bilateral epicanthic fold, hypertelorism, small autosomal genes, but only two studies11,12 have directly and low-set ears (total ear length 4.7 cm, o3rd centile), measured the spread of X inactivation through autosomal high nasal bridge, thin lips, microstomia and short neck. DNA by gene expression analysis. White et al11 found that Her hands were of normal length, but with tapering the spread of inactivation in an X;4 translocation was fingers, brachydactyly of the fifth fingers and hypoplasic discontinuous, while Sharp et al12 showed in five X;auto- creases. Mild genu valgum was present and all joints were some translocations that spread of gene silencing occurred mildly stiff. in either a continuous or discontinuous fashion in At the age of 10 years, she developed polyarticular different cases. juvenile arthritis, mainly involving hand and knee joints. We have used gene expression and CpG island methyla- Disease onset was insidious with morning stiffness and tion analysis in somatic cell hybrid clones to assess the arthralgia during the day. Rheumatoid factor was negative inactivation status of several chromosome 5 genes translo- and immunity-autoimmunity markers were within the cated onto the X chromosome in a girl with microcephaly, normal range. developmental delay and subtle dysmorphic features At last follow-up visit, at 11 years, she was 151 cm tall carrying an unbalanced 46, X, der(X)t(X;5)(q;q31) karyo- (90th–97th centile), weighed 56 Kg (4971 centile) and had type. a head circumference of 50 cm (3rd centile). Fat distribu- tion was predominantly truncal. All standard laboratory parameters were within the normal range and, despite an Materials and methods extensive clinical and ultrasound examination, no further Case report anomalies were found. Karyotype analysis revealed an The female proband was the third child of healthy unbalanced (X;5) translocation. unrelated Caucasian parents, ages 37 (mother) and 41 (father) at the time of her birth. Her older brother and sister Cytogenetic studies and Array-CGH were healthy. Chromosome analysis was performed on the proband’s and The family history is unremarkable. No drug, infection or parents’ blood, using standard high-resolution techni- radiation exposure during pregnancy is reported. The ques.13 The TelVysion kit with telomere-specific probes pregnancy was complicated by fetal chronic distress during was used according to the supplier’s instructions (Vysis the third trimester. Fetal bradycardia and cord prolapse in Inc., Downers Grove, IL, USA). the presence of intact membranes necessitated an emer- Molecular karyotyping was performed through Array- gency C-section at 39 weeks. At birth, she weighed 2410 g CGH with Agilent B100 Kb oligo-chip (Agilent Technologies, (o3rd centile), was 47 cm long (3rd centile) and her head Palo Alto, CA, USA; www.chem.agilent.com). The Agilent circumference was 31.5 cm (o3rd centile). APGAR scores kit platform is a 60-mer oligonucleotide-based microarray were 5/7/8 at 1, 5 and 10 min, respectively. In the neonatal that allows genome-wide survey and molecular profiling of period she was diagnosed with a small muscular ventricular genomic aberrations with a resolution of B100 kb (kit septal defect (VSD), which spontaneously closed within the 44B). DNAs (500 ng) of the patient and controls of the same first year of life. At 20 months, skull X-rays confirmed sex (Promega Co., Fitchburg, WI, USA) were double- microcephaly and did not reveal premature fusion of the digested with RsaI and AluI for 2 h at 371C. After column cranial sutures. At 2 1/2 years, a CT scan of the head purification, 2 mg of each digested sample were labelled showed no structural anomalies of the brain, normal by random priming (Invitrogen, Carlsbad, CA, USA) for corpus callosum, ventricles and basilar cisterns. At 5 1/2 2 h using Cy5-dUTP for the patient’s/ parents’ DNA and years, a cerebral MRI was normal. Cy3-dUTP for the control DNA. Labelled products were Audiological assessment at 4 and 6 years of age showed column purified. After probes denaturation and pre- mild bilateral conduction hearing loss (speech reception annealing with 50 mg of Cot-1 DNA, hybridisation was threshold of 30 dB) attributed to recurrent acute otitis performed at 651C with rotation for 40 h. After two media. Ophthalmologic evaluation showed hypermetropic washing steps, the array was analysed through the Agilent astigmatism OS4OD and left esophoria. scanner and Feature Extraction software (v8.1). A graphical Her psychomotor development was moderately delayed, overview was obtained using the CGH analytics software and in particular she displayed very short attention span (v3.4.4). European Journal of Human Genetics Spreading of X inactivation in a t(X;5) R Giorda et al 899 Somatic cell hybrid generation and molecular analysis Repeat distribution analysis Somatic cell hybrid clones were generated fusing the HPRT- We retrieved all the repeats and genes data for chromo- negative RJK88 Chinese hamster cell line with lympho- some 5 from the UCSC genome browser (http://geno- blastoid cell line from the patient.14 Clones were tested for me.ucsc.edu/) using the latest assembly (hg18). This the presence of der(X) or chromosome 5 by PCR with resulted in a total of 209531 repeats features. The polymorphic markers. frequencies of Alu, MIR, LINE and L1 elements have been Genomic DNA was extracted from the proband’s lym- calculated starting from 55 MB in nonoverlapping win- phoblastoid cell line and her parent’s blood using standard dows of 100 Kb.