PhD Pascale Guicheney
email: pascale.guicheney@upmc.fr

Nigel F Clarke

MS Svetlana Maugenre

PhD Aurélie Vandebrouck

Andoni Urtizberea

PhD Tobias Willer

PhD Rachel Peat

MD, PhD Françoise Gray

PhD Céline Bouchet

PhD Hiroshi Manya

PhD M B Chb

MD J Andoni Urtizberea

PhD Sandrine Vuillaumier-Barrot

PhD Tamao Endo

PhD Eliane Chouery

PhD Kevin P Campbell

MD, PhD André Mégarbané

Clarke Nf

Congenital Muscular Dystrophy type 1D (MDC1D) due to a large intragenic insertion/deletion involving intron 10 of the gene

Keywords: congenital muscular dystrophy 1D, alpha-dystroglycan, DNA duplication, muscle-eye-brain disease

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Alpha-dystroglycan (αDG) is a highly glycosylated cell surface protein that plays important roles in neuronal cell migration and cell-to-extracellular matrix interactions in muscle.

Abnormalities in the glycosylation of αDG is the hallmark histological abnormality and likely pathogenic mechanism for a group of congenital muscular dystrophies (CMD), collectively called the 'α-dystroglycanopathies'. [START_REF] Martin | Mechanisms of disease: congenital muscular dystrophies-glycosylation takes center stage[END_REF] At present, six genes have been linked to this common clinicopathological presentation (POMT1, POMT2, POMGNT1, FKTN, FKRP and LARGE). All of these encode confirmed or putative enzymes that are thought to function in the O-mannosyl glycosylation pathway. Abnormal αDG glycosylation correlates with an increasingly severe pattern of tissue effects that extends from an adult-onset limb-girdle muscular dystrophy with normal intelligence (LGMD type 2I) to severe congenital brain and eye malformations (Walker-Warburg syndrome, WWS). [START_REF] Martin | Mechanisms of disease: congenital muscular dystrophies-glycosylation takes center stage[END_REF] The LARGE gene became a candidate for human CMD after the discovery of the myodystrophic (myd) mouse that has a spontaneous null mutation in Large due to a genomic deletion involving exons 5 to 7. [START_REF] Lane | Myodystrophy, a new myopathy on chromosome 8 of the mouse[END_REF][START_REF] Mathews | Phenotypic and pathologic evaluation of the myd mouse. A candidate model for facioscapulohumeral dystrophy[END_REF][START_REF] Grewal | Mutant glycosyltransferase and altered glycosylation of alpha-dystroglycan in the myodystrophy mouse[END_REF] Another spontaneous murine mutant, named veils (vls) also results from an intragenic deletion leading to loss of exons 3 to 5 and premature protein termination. [START_REF] Lee | Ocular abnormalities in Large(myd) and Large(vls) mice, spontaneous models for muscle, eye, and brain diseases[END_REF] The two strains share typical cerebral, ocular and muscle changes of an α-dystroglycanopathy. MDC1D due to mutations in LARGE is the rarest of the α-dystroglycanopathies identified to date, with only three confirmed families previously described. [START_REF] Longman | Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan[END_REF][START_REF] Van Reeuwijk | Intragenic deletion in the LARGE gene causes Walker-Warburg syndrome[END_REF][START_REF] Mercuri | Congenital muscular dystrophies with defective glycosylation of dystroglycan: a population study[END_REF] Affected children have had typical neurological and muscle abnormalities associated with the α-dystroglycanopathies, but with very different severities; one family had mild muscle-eye-brain disease (MEB) and the other two had typical WWS. Another WWS patient has been reported with a single heterozygous nonsense mutation in LARGE. [START_REF] Godfrey | Refining genotype phenotype correlations in muscular dystrophies with defective glycosylation of dystroglycan[END_REF] Confirmation that this patient has CMD due to LARGE awaits the discovery of a second mutation. In this paper, we describe the fourth confirmed family with MDC1D due to a homozygous LARGE mutation, in which two sisters from Clarke NF 5 consanguineous parents have moderate mental retardation, cerebellar and pontine hypoplasia and persistent white matter abnormalities on brain MRI, features consistent with mild MEB.

We identified an unusual genetic mechanism for LARGE gene disruption, namely the splicing of an abnormal sequence into the LARGE mRNA transcript likely due to a duplication/deletion event involving intron 10. This report extends our knowledge of the clinical phenotype associated with LARGE, and is the second family where disease results from a large scale gene rearrangement.

The POMT1, POMT2 and POMGNT1 genes encode enzymes that together mediate the first two steps in the O-mannosyl glycosylation pathway. A recent study suggests that LARGE participates in post-phosphoryl glycosylation of a novel phosphorylated O-mannosyl glycan on the mucin-like domain of recombinant αDG, which is required for laminin binding. [START_REF] Yoshida-Moriguchi | O-mannosyl phosphorylation of alphadystroglycan is required for laminin binding[END_REF] Evidence from cell culture and a fukutin transgenic mouse suggests that fukutin physically interacts with POMGnT1 and can cause reduced POMGnT1 activity when abnormal. [START_REF] Xiong | Molecular interaction between fukutin and POMGnT1 in the glycosylation pathway of alpha-dystroglycan[END_REF] To investigate whether LARGE may also act by influencing the first steps of the O-mannosyl pathway, we assessed the activity levels of POMT and POMGnT1 in cultured lymphoblasts from one of our patients.

Case reports

The two affected girls are the only children of first-cousin parents from Lebanon. There is no other family history of neuromuscular disease. Both children were born full-term after uneventful pregnancies with normal fetal movements and normal deliveries. Birth weights, lengths and head circumferences were within normal limits.

Clarke NF 6

Patient 1: The older sibling first presented to medical attention at age 2 ½ months with hypotonia and regurgitation. Acquisition of all motor skills was delayed. She sat at age 9 months, stood unsupported at age 2½ years and at age 8 years she could climb stairs with difficulty but could not run. Her full scale IQ was 61. On examination at age 8 years, she weighed 24 kg (50 th centile), her height was 122.5 cm (35 th centile) and the occipitofrontal circumference (OFC) was 53 cm (75 th centile). Her speech was restricted to dysarthric, simple words and she followed simple commands. She had a myopathic face with an openmouth posture, and mild tongue hypertrophy. There was generalised muscle hypertrophy, most prominent in the calves, thighs and shoulder girdle. This contrasted with moderate proximal lower limb weakness that manifested as a waddling gait and a positive Gowers' manoeuvre. Upper limb strength was within normal limits. There were no joint contractures, except for mild tightness of the Achilles' tendons, associated with toe-walking. The only abnormalities found on eye assessment were mild myopia and strabismus. There was no history of seizures. Cardiac and respiratory examinations were normal. Brain Magnetic Resonance Imaging (MRI) performed at ages 3.5 and 6 years showed marked cerebellar atrophy (particularly affecting the vermis) and cerebellar cysts (figure 1). There was mild generalized cerebral and pontine atrophy, dilatation of all ventricular spaces and pachygyria of the frontal lobes (figure 1). Diffuse symmetrical high signal abnormalities were seen within the periventricular and deep white matter of both cerebral hemispheres, with sparing of the subcortical U fibres. These hyperintensities were unchanged at age 6 years. A generalized skeletal X-ray survey, abdominal ultrasound, echocardiogram, electroencephalogram, and auditory evoked potentials with brainstem responses were all normal. Creatine kinase (CK) levels were 30 times higher than the upper limit of normal.

Patient 2: The younger sister followed a similar clinical course to her sister but was less delayed in her motor and cognitive skills. She stood unsupported at age 16 months and Clarke NF 7 walked independently at age 2 years. On examination at age 4½ years, her weight was 15 kg (25 th centile), height 106 cm (75 th centile) and OFC 51 cm (50 th centile). There was generalised hypotonia, firm hypertrophic calves and a mild equinovarus foot position. Mild myopia was the only abnormality on eye examination. Myopathic features were noted on EMG while nerve conduction studies were normal. Brain MRI performed at 2 years showed diffuse high signal white matter abnormalities mainly in the frontal and parieto-occipital regions, dilated ventricular spaces and marked cerebellar hypoplasia. CK levels were 60 times the upper limit of normal.

Muscle biopsy findings

Muscle and skin biopsies were taken from Patient 1 from the right gastrocnemius muscle.

Standard histochemical analysis revealed a dystrophic pattern characterised by fibre size variation, markedly increased connective tissue, internalised nuclei and scattered necrotic fibres. Immunohistochemistry (IHC) for αDG showed markedly reduced staining using VIA4-1 (Millipore, Billerica, MA, USA), an antibody that recognizes a glycosylated epitope.

Laminin α2 expression was mildly reduced by IHC using antibodies that recognize both the 80 kDa (clone 5H2; Millipore, Billerica, MA, USA) and the 300 kDa (NCL-merosin;

Novocastra Labs, Newcastle, UK) fragments. Staining for β-dystroglycan (βDG), dystrophin, collagen VI, and sarcoglycans (α and γ) was normal.

Western blot analysis

Western blotting was used to investigate the degree of abnormal αDG glycosylation using a skin fibroblast cell line from Patient 1 as frozen muscle was not available. Fibroblasts were maintained at 37°C and 5% CO 2 in DMEM medium plus 20% fetal bovine serum, 0.5% penicillin-streptomycin (Gibco). Cultured cells were lysed and glycoproteins from cell Clarke NF 8 homogenates were enriched with WGA lectin as previously described. [START_REF] Xiong | Molecular interaction between fukutin and POMGnT1 in the glycosylation pathway of alpha-dystroglycan[END_REF] We performed immunoblots (for αDG and βDG) and laminin ligand overlay assays using polyvinylidene difluoride membranes and blots were developed by horseradish peroxidase (HRP) enhanced chemiluminescence (Pierce). [START_REF] Michele | Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies[END_REF] The monoclonal antibodies IIH6 and VIA4-1, that are specific for functionally glycosylated αDG, [START_REF] Ervasti | Membrane organization of the dystrophin-glycoprotein complex[END_REF] showed no detectable binding affinity to αDG from patient fibroblast cells even though βDG (monoclonal antibody 7D11) was normally present, consistent with a glycosylation defect in αDG (figure 2). This defect in αDG glycosylation also resulted in loss of laminin-1 ligand binding (figure 2).

Genetic analysis

We performed linkage analysis using microsatellite markers to the five α-dystroglycanopathy loci known at the time (POMGNT1, FKRP, POMT1, POMT2 and FKTN) and the DAG1 gene that encodes DG but we identified no homozygous linkage. A genome-wide linkage study using 400 microsatellite markers showed potential linkage to the newly identified LARGE locus and analysis of further markers supported homozygous linkage (figure 3). We sequenced the coding regions of LARGE bi-directionally by standard techniques.

Oligonucleotide primers at least 80 bp outside the exons were chosen for PCR amplication and sequencing on ABI3730 capillary electrophoresis system (Applied Biosystems), but no abnormalities were found. Subsequently mRNA was extracted from cultured patient EBVtransformed lymphoblasts from Patient 1 (Trizol method), cDNA was generated using oligodT Superscript II First-Strand Synthesis (Invitrogen, Carlsbad, CA, USA) and the LARGE gene transcript was sequenced using 14 overlapping primer pairs. Two abnormally large PCR products, together with a faint product of the expected size were obtained for primers that spanned exons 9 and 12 (see figure 4A). Sequencing revealed that most mRNA transcripts contained an abnormal 197 base pair (bp) sequence inserted between exons 10 and Clarke NF 9

11. Low levels of a 171 bp insertion (identical to the 197 bp sequence except lacking 26 bases from the 3` end) and a normally spliced transcript were also obtained. Identical splicing abnormalities were also seen in mRNA from cultured patient fibroblasts, except a greater proportion of transcripts showed normal splicing (figure 4A). The abnormal sequences mapped to part of a spliced EST (GenBank Accession No: DA935254) of unknown function, originally identified in a human splenic cDNA library, [START_REF] Kimura | Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes[END_REF] normally located 100 kb centromeric to the LARGE gene on chromosome 22q12.3 (figure 5). The presence of the two most abundant abnormal inserted sequences are predicted to immediately introduce a premature stop codon in the LARGE mRNA transcript, resulting in truncation of the LARGE protein mid-way through translation (figure 4B). Analysis of the inserted EST sequence identified potential splice acceptor and donor sites for both the larger and smaller insertions that likely mediate the abnormal splicing (E-figure 1). Archived frozen muscle was no longer available to verify the abnormalities in LARGE splicing in muscle.

To investigate whether the abnormal LARGE splicing arose due to a chromosomal duplication, we performed quantitative PCR (qPCR) analysis of the EST sequence from patient genomic DNA (gDNA) using the LightCycler® 480 (Roche Diagnostics, Basel, Switzerland) and the SYBR Green I detection system. We normalised gDNA concentrations between individual samples using PCR primers for a distant gene (PTPLA on chromosome 10). E-Table 1 lists the key primer pairs used. The EST (DA935254) was present in double the expected copy number in both patients compared with two healthy unrelated individuals (figure 4C), consistent with a homozygous duplication of the EST sequence in both patients (ie they have four copies instead of two). To investigate the size of the putative duplication, we repeated qPCR analysis for genomic regions at variable distances up-and downstream of the EST sequence (E-Table 1). These studies indicate that between 40825 and 42937 bp of Clarke NF 10 genomic DNA surrounding the EST sequence is present at twice normal levels in gDNA in both patients (figure 4C). Both parents had 1.5 times the normal copy number, consistent with each having one copy of the chromosome 22 duplication and a normal allele. The 3` breakpoint is likely located in a 1313 bp region between regions 3b and 3c (Chr 22:33548218-33549530; GRCh37/hg19 assembly) and the 5` breakpoint in a 799 bp region between regions 5b and 5c (Chr 22:33590356-33591154).

Together, these results indicate that the patients are homozygous for a duplication of gDNA around EST (DA935254) and the most likely site of insertion for the duplicated DNA is intron 10 of LARGE, leading to the abnormal splicing of the EST into the LARGE mRNA transcript between exons 10 and 11.

To identify the site of the insertion in intron 10 of LARGE, we designed twelve pairs of overlapping primers (E-figure 2) to cover the whole length of the intron (44117 bp). With two primer pairs (5aF-5aR, 6aF-6aR) we amplified the expected bands in controls and parents but not in the two patients indicating that the insertion should be in this region (E-figure 2). We repeated the same strategy with several other primer pairs chosen inside this region and in the boarding regions to finally determine a region of 3012 to 4175 bp that could never be amplified in the patients, which likely corresponds to a deleted region associated with the insertion. By qPCR analysis using primer pairs L5F-L6R and L6F and L7R (Supplemental E-Table 1), both parents had reduced amplification compared to controls, consistent with them being heterozygous for a deletion in this region. We were not able to amplify any product with primers chosen on the borders of the insertion and on the non deleted sides of the intron, and thus did not identify the exact breakpoints for the insertion/deletion rearrangement.

Clarke NF 11

As repetitive sequences are implicated in the pathogenesis of recurrent chromosomal deletion or duplication syndromes (eg. CMT1A) [START_REF] Pentao | Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit[END_REF] we analysed intron 10 and the duplicated region around EST DA935254 for homologous sequences. Both regions shared multiple SINE, LINE and LTR repeats that may have played roles in the genesis of the mutation, but no extended regions of homology.

POMT and POMGnT1 activity in patient lymphoblasts

The POMT and POMGnT1 enzymes mediate the first and second steps respectively of the Omannosyl glycosylation of αDG. To investigate whether LARGE mutations may disrupt the glycosylation of αDG by influencing the activity of either POMTs or POMGnT1, we assessed the enzymatic activity of these enzymes in cultured patient lymphoblasts using previously validated assays. [START_REF] Manya | Protein O-mannosyltransferase activities in lymphoblasts from patients with alpha-dystroglycanopathies[END_REF] The POMT activity was based on the amount of [ 3 H]mannose transferred from [ 3 H]mannosylphosphoryldolichol to a glutathione-S-transferase fusion αDG, and the POMGnT1 activity was based on the amount of [ 3 H]GlcNAc transferred from UDP-[ 3 H]GlcNAc to a mannosylpeptide. In brief, we incubated microsomal membrane fractions prepared from patient and control EBV-immortalised B lymphocytes with an acceptor substrate and radio-labelled sugar donor substrate. We then assessed the incorporation of radioactivity into each acceptor substrate. We normalised control and patient samples using the activity level of GnT1, a Golgi enzyme involved in the N-glycosylation pathway, which functions independently of O-glycosylation. Results showed no difference between the patient and eight control samples for POMT/GnT1 activity (patient 0.283 vs control 0.287 ± 0.067 standard deviation) or for POMGnT1/GnT1 activity (patient 0.068 vs control 0.071 ± 0.019).

Discussion

Clarke NF 12 In this paper we report the fourth confirmed family with MDC1D due to mutations in LARGE. The clinical abnormalities are typical for the α-dystroglycanopathy group of disorders. Both sisters have typical features of an alpha-dystroglycanopathy, namely raised CK levels, dystrophic changes on muscle histology associated with reduced glycosylated alpha-dystroglycan staining, intellectual disability and brain malformations. In terms of severity of neurological involvement, the two sisters are best classified as having mild MEB disease, even though there were only minor ocular abnormalities. The findings were similar to the first family identified with mutations in LARGE with mild/moderate developmental abnormalities of the cerebellum, pons, brainstem and cerebrum although our patients are the first reported with cerebellar cysts. [START_REF] Longman | Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan[END_REF] Even though standard sequencing of the coding regions of LARGE was normal in the proband of this family, we persisted with LARGE analysis because there was homozygous linkage to the gene locus in the context of consanguinity. Analysis of cDNA, generated from mRNA derived from patient lymphoblasts, showed an abnormally large product for a PCR reaction that spanned exons 9 to 12. Sequencing of this product showed that an abnormal sequence of either 197 or 171 bases was spliced between exons 10 and 11 in almost all transcripts. The abnormal sequence is normally situated on chromosome 22 centromeric to the 3` end of LARGE. In this position, it would not usually be incorporated into the LARGE transcript, even though the sequence contains the necessary splice donor and acceptor sites. To explain its incorporation into the LARGE mRNA, we hypothesised that the EST sequence was translocated into intron 10 of LARGE in our family, possibly through duplication of this genomic region. We assessed the copy number of the EST sequence by qPCR relative to other genes in patient genomic DNA and showed twice the expected levels, consistent with a genomic duplication in a homozygous state. Both parents had a 1.5-fold increase in copy Clarke NF 13 number (an increase from two to three copies of the sequence), consistent with both of them being asymptomatic heterozygous carriers of the duplication. To map the approximate size of the duplication, we repeated qPCR analysis up-and downstream of the EST sequence and showed the duplication spans between 40 and 43 kb of genomic DNA. Even though we identified the likely site of insertion in the central part of intron 10 and delimited a large intronic region that cannot be amplified in the patients, mapping the precise breakpoints of the proposed duplication/insertion was not feasible, possibly due to the presence of a more complex rearrangement than anticipated.

Analysis of LARGE mRNA transcripts from patient lymphoblasts and fibroblasts suggests that most LARGE mRNA transcripts contain an abnormal sequence. Both versions of the abnormal insertion are predicted to immediately introduce a premature stop codon mid-way through protein translation, just before the second catalytic domain, and result in a nonfunctional protein (figure 4B). Small amounts of normally spliced transcript were also seen and the levels appeared slightly higher in fibroblasts than lymphoblasts, suggesting this may vary in different tissues. Unfortunately it was not possible to assess levels of normally spliced LARGE mRNA or protein function either in muscle or brain, the two main tissues that show pathology in the α-dystroglycanopathies. We observed no functionally glycosylated αDG by Western blot analysis in patient fibroblasts but it is likely that low levels of functional αDG are present in patient tissues at levels below the threshold of detection by Western blot for several reasons. Residual glycosylated αDG staining was present in the muscle biopsy and both sisters have less severe cognitive deficits and brain malformations than a patient with homozygous null mutations in LARGE, who had a WWS phenotype. [START_REF] Van Reeuwijk | Intragenic deletion in the LARGE gene causes Walker-Warburg syndrome[END_REF] We also detected low levels of a normally spliced LARGE gene mRNA transcript in both lymphoblasts and fibroblasts, which should allow normal LARGE protein to be produced at low levels.

Clarke NF 14

We also investigated whether LARGE may exert some of its effects on αDG glycosylation through interactions with either the POMT or POMGnT1 enzymes. Both POMT and POMGnT1 activities were normal in patient lymphoblasts, strongly suggesting that the two initial steps of αDG glycosylation occur independently of LARGE.

At present it appears that LARGE is a rare cause of CMD since the coding regions of the gene have been screened in large cohorts of patients with α-dystroglycanopathy of unknown cause. [START_REF] Longman | Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of alpha-dystroglycan[END_REF][START_REF] Mercuri | Congenital muscular dystrophies with defective glycosylation of dystroglycan: a population study[END_REF] Although experience is very limited at present, it is notable that two of the four confirmed families with CMD due to LARGE mutation reported have had large gene rearrangements, either a deletion involving several exons 7 or a likely intragenic insertion/deletion (our family). In another family a heterozygous nonsense mutation (p.Trp516X) was identified on standard gene sequencing, leaving open the possibility that a large deletion/duplication or rearrangement on the other allele was overlooked. [START_REF] Godfrey | Refining genotype phenotype correlations in muscular dystrophies with defective glycosylation of dystroglycan[END_REF] In addition, consanguineous families are described that link to the LARGE locus but no mutation has been found by exon sequencing. [START_REF] Van Reeuwijk | Intragenic deletion in the LARGE gene causes Walker-Warburg syndrome[END_REF][START_REF] Manzini | Ethnically diverse causes of Walker-Warburg syndrome (WWS): FCMD mutations are a more common cause of WWS outside of the Middle East[END_REF] LARGE was so named for the enormous genomic region it occupies on chromosome 22q12.3 (647 kb), due to the presence of very large introns enriched in low copy repeats (LCRs). The structure of the LARGE gene may predispose to mutations that involve duplication or deletion of large regions of DNA due to non-allelic homologous recombination between LCRs, the proposed mechanism for many genomic disorders. [START_REF] Inoue | Molecular mechanisms for genomic disorders[END_REF] Sequencing of coding regions from genomic DNA will not identify many large gene rearrangements. Therefore, sequencing LARGE from cDNA and the application of CGH, qPCR, and other methods for detecting rearrangement mutations should be considered routinely.

Clarke NF 15

Acknowledgements

We thank Eric Villard for helpful discussion. NC was supported by a joint grant from the Australian National Health and Medical Research Council (NHMRC; ID 402861) and the Institut National de la Santé et de la Recherche Médicale, AV and RP by Marie Curie grants from the European commission (OIF N°039762 and IIF N°221685). This work was also supported by research grants from the Ministry of Health, Labour and Welfare of Japan (HM, TE; for Nervous and Mental Disorders (20A-13) and Research on Psychiatric and Neurological Diseases and Mental Health), from The Takeda Science Foundation (HM, TE) and from the Paul D. Wellstone Muscular Dystrophy Cooperative Research Center grant (1U54NS053672; KPC). KPC is an Investigator of the Howard Hughes Medical Institute. Clarke NF 16

Title for figure 2 Western blot analysis of α-dystroglycan Legend for figure 2 Immunoblot analysis of WGA-enriched homogenates from fibroblasts from Patient 1 and a healthy individual (control). Absent staining of antibodies IIH6 and VIA4 that bind to glycosyl epitopes on α-dystroglycan (α-DG) and a laminin-1 (Lam) overlay assay show abnormalities in αDG glycosylation in patient fibroblasts that is associated with loss of laminin-1 protein binding. Beta-dystroglycan (β-DG) is used as a loading control. Title for figure 3 Genotyping for the LARGE locus Legend for figure 3 Clarke NF 21

Family tree showing analysis of microsatellite markers in close proximity to the LARGE locus (22q12.3). Both affected children are homozygous for four contiguous markers, consistent with homozygosity by descent for this chromosomal region. Title for figure 4 Analysis of the LARGE gene insertion in genomic and cDNA Legend for figure 4 A; Agarose gel showing the PCR products from patient (P) and control (C) cDNA generated from cultured lymphoblasts (Lymph) and fibroblasts (Fib) using primers that span exons 9 to 12 of LARGE (see E-Table 1). In patient cDNA, two abnormally high bands predominate that correspond to insertions of abnormal DNA sequences between exons 10 and 11. A faint band of the normal size is also seen (arrow). B; Diagram showing the amino acid sequence that is coded by normal and patient cDNA around the exon10/11 boundary. The abnormal insertion found in patient cDNA results in the introduction of a premature STOP codon, predicted to cause protein termination mid-way through the translation of the LARGE protein. C; Results from qPCR analysis of genomic DNA (gDNA) for EST DA935254 and the surrounding chromosomal region. gDNA concentrations between samples were adjusted using qPCR results from a distant gene (PTPLA) and results were scaled so that control samples had a mean copy number of 2 at each point (data not shown). In patient gDNA, qPCR analysis of the EST sequence and for five regions at variable distances upstream and downstream shows twice the expected copy number, consistent with homozygous duplication of this region.

Genomic copy number returns to normal for markers 5b and 3b in patient gDNA, and therefore these markers define the maximum size of the duplication. Analysis of gDNA from both parents shows that both had three copies of the duplicated region, consistent with each


Valeria Romanelli

Heloisa N M Meneses

Luis Fernández

Victor Martínez- Glez

Ricardo Gracia-Bouthelier

Mario Fraga

Encarna Guillén

Julián Nevado

Esther Gean

Loreto Martorell

Victoria Esteban Marfil

Sixto García-Miñaur

Pablo Lapunzina
email: plapunzina.hulp@salud.madrid.org

Beckwith Wiedemann syndrome and Uniparental Disomy 11p: Fine mapping of the recombination breakpoints and evaluation of several techniques

Keywords: Beckwith-Wiedemann syndrome, uniparental disomy, breakpoint, SNParray

Beckwith-Wiedemann syndrome (BWS) is a phenotypically and genotypically heterogeneous overgrowth syndrome characterized by somatic overgrowth, macroglossia and abdominal wall defects. Other usual findings are hemihyperplasia, embryonal tumors, adrenocortical cytomegaly, ear anomalies, visceromegaly, renal abnormalities, neonatal hypoglycaemia, cleft palate, polydactyly and a positive family history.

BWS is a complex, multigenic disorder associated, in up to 90% of patients, with alteration in the expression or function of one or more genes in the 11p15.5 imprinted gene cluster. There are several molecular anomalies associated with BWS and the large proportion of cases, about 85%, is sporadic and karyotypically normal. One of the mayor categories of BWS molecular alteration (10-20% of cases) is represented by mosaic paternal uniparental disomy (pUPD11), namely patients with two paternally derived copies of chromosome 11p15 and no maternal contribution for that. In these patients, in addition to the effects of IGF2 overexpression, a decreased level of the maternally expressed gene CDKN1C may contribute to the BWS phenotype.

In this paper, we reviewed a series of 9 patients with BWS due to pUPD11 using several methods with the aim to evaluate the percentage of mosaicism, the methylation status at both loci, the extension of the pUPD11 at the short arm and the breakpoints of recombination. Fine mapping of mitotic recombination breakpoints by SNP-array in individuals with UPD and fine estimation of epigenetic defects will provide a basis for understanding the aetiology of BWS, allowing more accurate prognostic predictions and facilitating management and surveillance of individuals with this disorder.

INTRODUCTION

Beckwith-Wiedemann syndrome [BWS (MIM 130650)] is a phenotypically and genotypically heterogeneous overgrowth syndrome characterized by somatic overgrowth, macroglossia and abdominal wall defects. Other usual findings are hemihyperplasia, embryonal tumors, adrenocortical cytomegaly, ear anomalies, visceromegaly, renal abnormalities, neonatal hypoglycaemia, cleft palate, polydactyly and a positive family history [START_REF] Beckwith | Extreme cytomegaly of the adrenal fetal cortex, omphalocele, hyperplasia of kidneys and pancreas, and Leydig-cell hyperplasia: Another syndrome?[END_REF][2][START_REF] Martinez | Clinical features in the Wiedemann-Beckwith syndrome[END_REF][START_REF] Wiedemann | Familal malformation complex with umbilical hernia and macroglossia: A "new syndrome[END_REF] .

BWS is a complex, multigenic disorder associated, in up to 90% of patients, with alteration in the expression or function of one or more genes in the 11p15.5 imprinted gene cluster [START_REF] Maher | Wiedemann syndrome: imprinting in clusters revisited[END_REF] (Figure 1a). There are several molecular anomalies associated with BWS and the large proportion of cases, about 85%, is sporadic and karyotypically normal.

Chromosomal rearrangements are relatively rare (~2-3% of cases) and comprise translocations or inversions (typically maternally inherited), microdeletions of KvDMR 6 or H19DMR [START_REF] Riccio | Inherited and Sporadic Epimutations at the IGF2-H19 locus in Beckwith-Wiedemann syndrome and Wilms' tumor[END_REF] (maternally inherited) and paternal duplications. Point mutations in CDKN1C, a cyclin-dependent kinase inhibitor acting as negative regulator of cell proliferation, have been found in 5-7% of sporadic BWS cases [START_REF] Lam | Analysis of germline CDKN1C (p57KIP2) mutations in familial and sporadic Beckwith-Wiedemann syndrome (BWS) provides a novel genotype-phenotype correlation[END_REF][START_REF] Lee | Low frequency of p57KIP2 mutation in Beckwith-Wiedemann syndrome[END_REF][START_REF] Romanelli | CDKN1C mutations in HELLP/preeclamptic mothers of Beckwith-Wiedemann Syndrome (BWS) patients[END_REF][START_REF] Romanelli | CDKN1C (p57(Kip2)) analysis in Beckwith-Wiedemann syndrome (BWS) patients: Genotype-phenotype correlations, novel mutations, and polymorphisms[END_REF] and in approximately 40% of cases with a positive family history [START_REF] O'keefe | Coding mutations in p57KIP2 are present in some cases of Beckwith-Wiedemann syndrome but are rare or absent in Wilms tumors[END_REF] .

The largest molecular subgroup, about 60% of cases, is represented by BWS patients that carry an epigenetic error on one or more members of 11p15.5 imprinted gene cluster. Two main epigenetic alterations in BWS patients have been described, one is represented by gain of methylation at the paternal H19DMR (observed in 5-10% of cases), and it is associated with loss of H19 expression and IGF2 biallelic expression [START_REF] Reik | Maher ER Allelic methylation of H19 and IGF2 in the Beckwith-Wiedemann syndrome[END_REF][START_REF] Schneid | Parental allele specific methylation of the human insulin-like growth factor II gene and Beckwith-Wiedemann syndrome[END_REF] . The other alteration is loss of maternal methylation of KvDMR (observed in ~50% of BWS patients), usually accompanied by biallelic expression of the KCNQ1OT1 transcript and down-regulation of CDKN1C [START_REF] Engel | Epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome[END_REF][START_REF] Lee | Loss of imprinting of a paternally expressed transcript, with antisense orientation to KVLQT1, occurs frequently in Beckwith-Wiedemann syndrome and is independent of insulin-like growth factor II imprinting[END_REF][START_REF] Smilinich | A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome[END_REF] . Finally, another large category of BWS molecular alteration, 10-20% of cases, is represented by paternal uniparental disomy (pUPD), namely patients with two paternally derived copies of chromosome 11p15 and no maternal contribution for that region [START_REF] Henry | Uniparental paternal disomy in a genetic cancer-predisposing syndrome[END_REF][START_REF] Slatter | Mosaic uniparental disomy in Beckwith-Wiedemann syndrome[END_REF] . In these patients, in addition to the effects of IGF2 overexpression, a decreased level of the maternally expressed gene CDKN1C may contribute to the BWS phenotype [START_REF] Lee | Low frequency of p57KIP2 mutation in Beckwith-Wiedemann syndrome[END_REF][START_REF] O'keefe | Coding mutations in p57KIP2 are present in some cases of Beckwith-Wiedemann syndrome but are rare or absent in Wilms tumors[END_REF][START_REF] Hatada | An imprinted gene p57KIP2 is mutated in Beckwith-Wiedemann syndrome[END_REF] .

It has been demonstrated that BWS patients with paternal UPD always show mosaicisms, suggesting that all cases had arisen as a postzygotic event [START_REF] Slatter | Mosaic uniparental disomy in Beckwith-Wiedemann syndrome[END_REF][START_REF] Catchpoole | Epigenetic modification and uniparental inheritance of H19 in Beckwith-Wiedemann syndrome[END_REF][START_REF] Henry | Somatic mosaicism for partial paternal isodisomy in Wiedemann-Beckwith syndrome: a post-fertilization event[END_REF] ; a possible explanation is that lack of one or more chromosome 11 maternally expressed genes may lead to embryonic lethality [START_REF] Cooper | Mitotic recombination and uniparental disomy in Beckwith-Wiedemann syndrome[END_REF] . It was also suggested that the degree of mosaicism and the location of the genetic abnormality in different tissue is strongly associated with the pathological phenotype [START_REF] Itoh | Proportion of cells with paternal 11p15 uniparental disomy correlates with organ enlargement in Wiedemannbeckwith syndrome[END_REF] . The extent of pUPD at chromosome 11p has been studied by means of STR markers; the critical region for pUPD is telomeric to chromosome 11p13 and always includes the region where map some BWS genes (IGF2, H19 and CDKN1C) [START_REF] Slatter | Mosaic uniparental disomy in Beckwith-Wiedemann syndrome[END_REF][START_REF] Catchpoole | Epigenetic modification and uniparental inheritance of H19 in Beckwith-Wiedemann syndrome[END_REF][START_REF] Cooper | Mitotic recombination and uniparental disomy in Beckwith-Wiedemann syndrome[END_REF] . Some cases with mosaic pUPD for the whole chromosome 11 have been also described and the clinical findings did not differ from patients with pUPD restricted to a small part of 11p [START_REF] Cooper | Mitotic recombination and uniparental disomy in Beckwith-Wiedemann syndrome[END_REF][START_REF] Dutly | Seven cases of Wiedmann-Beckwith syndrome, including the first reported case of mosaic paternal isodisomy along the whole chromosome 11[END_REF] . While the extent of segmental disomy and proportion of cells with pUPD is variable, in all BWS cases the paternal UPD is isodisomic. This suggests that cells with paternal UPD of chromosome 11 may have a selective growth advantage and that maternal UPD cells die or there is a partition to different parts of the conceptus [START_REF] Bischoff | Single cell analysis demonstrating somatic mosaicism involving 11p in a patient with paternal isodisomy and Beckwith-Wiedemann syndrome[END_REF] .

Clinically, a clear association between mosaic UPD and hemihyperplasia exists [START_REF] Engel | Epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome[END_REF][START_REF] Slatter | Mosaic uniparental disomy in Beckwith-Wiedemann syndrome[END_REF][START_REF] Henry | Somatic mosaicism for partial paternal isodisomy in Wiedemann-Beckwith syndrome: a post-fertilization event[END_REF][START_REF] Cooper | Molecular subtypes and phenotypic expression of Beckwith-Wiedemann syndrome[END_REF] . In addition, it was noted that neoplasias and Wilms' tumors are more frequent in BWS patients with pUPD or H19DMR hypermethylation than in BWS patients with other molecular defect [START_REF] Cooper | Molecular subtypes and phenotypic expression of Beckwith-Wiedemann syndrome[END_REF][START_REF] Bliek | Increased tumour risk for BWS patients correlates with aberrant H19 and not KCNQ1OT1 methylation: occurrence of KCNQ1OT1 hypomethylation in familial cases of BWS[END_REF][START_REF] Debaun | Feinberg AP Epigenetic alterations of H19 and LIT1 distinguish patients with Beckwith-Wiedemann syndrome with cancer and birth defects[END_REF] . Moreover, it has been hypothesized that extremely high levels of UPD might drive severe phenotypic expression of BWS [START_REF] Smith | Severe presentation of Beckwith-Wiedemann syndrome associated with high levels of constitutional paternal uniparental disomy for chromosome 11p15[END_REF] ; but it is often difficult to determine if levels of UPD correlate with severity of the phenotype especially when the tissues/organs involved are not usually directly tested. Finally, in view of the evidence of imprinted transcripts at the Wilms' tumour suppressor gene (WT1 at 11p13) [START_REF] Malik | Identification of differential methylation of the WT1 antisense regulatory region and relaxation of imprinting in Wilms' tumor[END_REF][START_REF] Malik | Identification of an antisense WT1 promoter in intron 1: implications for WT1 gene regulation[END_REF][START_REF] Ward | Regulation of the Wilms' tumour suppressor (WT1) gene by an antisense RNA: a link with genomic imprinting?[END_REF] , it is interesting to evaluate whether disomy extended to WT1 influenced the risk of neoplasia (Figure 1a).

In this paper, we evaluated a series of 9 patients with BWS due to patUPD11p using several methods with the aim to evaluate the percentage of mosaicism, the methylation status at both loci, the extent of the pUPD at the short arm and their breakpoints. Fine mapping of mitotic recombination breakpoints by SNP-array in UPD and fine estimation of epigenetic defects will provide a basis for understanding the aetiology of BWS. In addition, it would allow more accurate prognostic predictions providing a better management and surveillance of BWS children.

PATIENTS AND METHODS

Patients

From a total of 132 patients with presumptive diagnosis of BWS included in the Spanish Overgrowth Syndrome Registry, 92 had confirmed molecular diagnosis of the disorder. Among these patients we detected 71 with hypomethylation at KvDMR, 8 with point mutation on CDKN1C gene, 2 with hipermethylation of H19DMR and 2 with paternal duplication of 11p15.5 region. Finally, nine out of these 92 patients had laboratory results indicative of pUPD11. Clinical data of BWS individuals including personal and family history, clinical, laboratory and X-rays, pedigree, and follow-up evolution were included in a database. Parental data were also recorded. The Institutional IRB at Hospital Universitario La Paz approved this study as part of the research study at the Spanish Overgrowth Syndrome Registry (# HULP-PI446).

Cytogenetics and DNA extraction

Karyotypes were performed by standard methods in all patients. A minimum of 20 cells were counted with a resolution of at least 550 bands. FISH analysis using the D11S2071 probe was applied to all patients. DNA was extracted from blood using Qiagen kits Puregene Blood Core Kit (Maryland, USA) in all patients and available parents.

STRs segregation analyses

A panel of 6 microsatellites mapping to the short arm of chromosome 11 was evaluated in patients and their parents (when available) (Figure 1b). We used the following STRs: TH01, D11S1318, D11S4088, D11S1338, D11S1346 and D11S4046.

Sequences of the primers were obtained from the NCBI public database (http://www.ncbi.nlm.nih.gov/sites/entrez). We calculated the percentage of mosaicism as recommended by Sasaki et al. [START_REF] Sasaki | Japanese and North American/European patients with Beckwith-Wiedemann syndrome have different frequencies of some epigenetic and genetic alterations[END_REF] . The calculation was as follows: (K-1)/(K+1) x 100;

where K is the ratio of the intensity of the parental alleles (paternal/maternal ratio) of the test sample.

Methylation-Specific MLPA (MS-MLPA)

MS-MLPA with 100 ng of genomic DNA, the SALSA ME030-B1 (11p15 region, BWS/SRS; MRC-Holland, Amsterdam, Holland) and the methylation-sensitive restriction enzyme HhaI (Promega Corporation) was performed according to the manufacturer's recommendations. MS-MLPA PCR products were analyzed on an ABI 3130 automated sequencer (Applied Biosystems, Foster City, California, USA). To calculate the gene dosage, the analysis of results was based on the comparison between the signals of undigested samples and undigested controls. Calculation of the methylation level of probes containing the HhaI restriction site was performed comparing the signals of restriction digested samples with undigested samples. The analysis of raw data was carried out using an Excel-based in-house program (Meth-HULP v1.1; available upon request). We normalized the raw value of peak areas in both controls and patient samples for undigested and digested samples and quantified gene dosage and methylation level for each sample.

Sodium bisulphite conversion and Methylation Specific-High Resolution Melting analysis (MS-HRMA)

Before MS-HRM assay, genomic DNA of controls and patients were treated with sodium bisulphite using the EZ DNA methylation Kit (Zymo research, Orange, CA, USA) according to the manufacturer's instructions. Bisulphite converts unmethylated cytosines to uracil, whereas methylated cytosines remain unreactive. Specific primers (available upon request) to amplify H19 and KvDMR from bisulphite-converted DNA were designed by means of Methylation Primer Express Software v1.0 (Applied Byosistems). The primers amplify both methylated and unmethylated template, according to the principles set out to compensate for PCR bias [START_REF] Wojdacz | Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay[END_REF] . Specificity of primers was confirmed by sequencing of the PCR products. PCR amplification and MS-HRM analysis were done in a LyghtCycler 480 Real-Time PCR System (Roche Diagnostics, Germany). Normalisation of the melting curves was carried out using the software provided with the LyghtCycler 480 (Gene Scanning software v1.5). To visually estimate the curves, we included two synthetic DNA with standard methylation of 0% and 100% as controls (EpiTect PCR Control DNA Set, Qiagen). We calculated the level of methylation by the MS-HRM assay, evaluating the area under the curve in the plateau phase. The position of the plateau at the vertical scale represents the ratio between the methylated and unmethylated allele.

Pyrosequencing of imprinted centres at 11p15

Quantitative analysis of methylation at the KvDMR and H19 imprinting centres was also performed by pyrosequencing. Specific primers (available upon request) to amplify and to pyrosequence the two amplicons were designed by means of Pyrosequencing Assay Design Sofware (Biotage, Charlottesville, VA). The forward primer for H19 and reverse primers for KvDMR amplification were 5'-biotinylated to facilitate single-strand DNA template isolation for the pyrosequencing reaction.

Preparation of the single-strand DNA template for pyrosequencing was performed using the PSQ Vacuum Prep tool (Biotage, Charlottesville, VA) according to the manufacturer's instruction. The biotinylated PCR product was immobilized on streptavidin-coated Sepharose high-performance beads (Amersham Biosciences, Piscataway, NJ) and processed to obtain single strand DNA using the PSQ 96 Sample Preparation Kit (Biotage) according to the manufacturer's instructions. The sequencingby-synthesis reaction of the complementary strand was automatically performed on a PSW 96MA instrument (Biotage) at room temperature using PyroGold reagents (Biotage). As nucleotides were dispensed, a light signal was generated proportional to the amount of each incorporated nucleotide. These light signals were detected by a charge-coupled device camera and converted to peaks in a sequencing program that was automatically generated in real time for each sample. The Pyro Q-CpG software (Biotage) automatically determines individual methylation frequencies for all CpG sites in the amplicon; the degree of methylation is calculated from the ratio of the peak heights of C and T.

High Density Single Nucleotide Polymorphism-arrays (SNP-arrays)

DNAs were extracted by routine methods and were quantified using PicoGreen (Invitrogen Corporation, Carlsbad, CA). A genome-wide scan of 620,901 tag SNPs was conducted on the patients, using the Illumina Human610-Quad BeadChip according to the manufacturer's specifications (Illumina, San Diego, CA). GenCall scores < 0.15 at any locus were considered "no calls". Image data was analyzed using the Chromosome Viewer tool contained in Beadstudio 3.2 (Illumina, San Diego, CA). The metric used was the log R ratio which is the log (base 2) ratio of the observed normalized R value for a SNP divided by the expected normalized R value 36 . In addition, an allele frequency analysis was applied for all SNPs. All genomic positions were based upon NCBI Build

(dbSNP version 126).

RESULTS

Paternal UPD and establishment of mosaicism percentage

All 9 cases of UPD have been diagnosed by analysis of STRs markers mapping at the short arm of chromosome 11, and have shown paternal isodisomy (Supplementary Figure 1). We calculated the percentage of mosaicism by means of STRs and SNP-arrays (Figure 2a). The calculation of mosaicism using STRs has been done as recommended by Sasaki et al. [START_REF] Sasaki | Japanese and North American/European patients with Beckwith-Wiedemann syndrome have different frequencies of some epigenetic and genetic alterations[END_REF] (Table Ia). Taking into account that isodisomy is paternal we could estimate the percentage of mosaicism in the SNP-array as the higher allele B frequency (Table II).

Correlation between the percentage of the UPD cells and the metylation index

As expected, the degree of mosaicism, calculated by SNP-array, and the level of methylation defect, estimated by pyrosequencing (Table Id), showed very high and statistically significant correlation: R 2 =+0.933 for H19DMR and R 2 =-0.833 for KvDMR(Figure 3a; Supplementary Table I).

Extent of the pUPD and correlation with methylation index and mosaicism

Fine mapping of mitotic recombination breakpoint in pUPD of chromosome 11 has been performed by SNP-array (Figure 2a,2b). To estimate the extent of pUPD we mapped the latest SNP in pUPD and the first SNP in heterozygosis and we evaluated the minimum and the maximum size of pUPD region for each patient (Table II). We calculated the methylation index in both imprinting centres of 11p15.5 region, H19 and KvDMR, through MS-MLPA, MS-HRM and pyrosequencing analyses (Supplementary Figure 1b,2a,2b; Table Ib, Ic, Id). We also performed a correlation analysis between the extent of the pUPD and the level of methylation (obtained by CpG Q-Pyro) and mosaicism (calculated by STR analysis). As we expected, there has been no correlation among these parameters (Figure 3b; Supplementary Table I).

Fine mapping of the breakpoints

Fine mapping of mitotic recombination breakpoints in pUPD of chromosome 11 by SNP-arrays did not provide strong evidence for recombination hot-spots. The locations of mitotic breakpoints were different in all patients (Figure 2b; Table II).

However, despite of variability of the extent of isodisomy, the region of paternal UPD invariably includes the whole imprinted gene cluster at 11p15.5 (Figure 2b).

DISCUSSION

Paternal UPD of chromosome 11p is a relatively common and cancer prone mechanism in BWS patients [START_REF] Cooper | Molecular subtypes and phenotypic expression of Beckwith-Wiedemann syndrome[END_REF] ; thus patients with this molecular subtype should be closely evaluated and prospectively followed-up. Regional or partial UPD are either caused by abnormal recombination between non-sister chromatids during mitosis or by deletion of a region and further duplication of the homologous region of the other chromosome.

From a total of 92 patients with confirmed molecular diagnosis of BWS only 9 patients (10%) had patUPD11p. This proportion is slightly lower than previous reports where close to 15-20% of the patients with BWS showed patUPD11p. This lower proportion may be due to the specific characteristics of our Registry with a high number of pregnancies obtained after assisted reproductive technologies, a procedure that it is hypothesized that predisposes to aberrant mehtylation of the centromeric imprinting center (IC2) [START_REF] Amor | A review of known imprinting syndromes and their association with assisted reproduction technologies[END_REF] .

Percentage of 11p mosaicism and BWS phenotype

It has been demonstrated that phenotypic expression of BWS correlates with elevated levels of UPD in the organs and tissues that were tested [START_REF] Itoh | Proportion of cells with paternal 11p15 uniparental disomy correlates with organ enlargement in Wiedemannbeckwith syndrome[END_REF] . However, it must be noted that the level of UPD has not been found to correlate with phenotypic expression. Our patients had clinical heterogeneity and lack of correlation with the percentage of mosaicism as well as the extension onto the short arm of chromosome 11. The only constant finding in all patients with patUPD11p in our series is hemihypertrophy. As an example of such lack of correlation, patient UPD 8 showed macrosomia at birth, hemihypertrophy and mild macroglossia and had 31 Mb of extension and 70 % of mosaicism and patient UPD 6 who has a smaller extension (6.6 Mb) and 64 % of mosaicism showed a more expanded phenotype including macrosomia, polyhydramnios, umbilical hernia, hemihypertrophy, macroglossia and hypoglycaemia (Supplementary Table II). Thus, larger size of mosaic UPD and/or higher percentage of mosaicism do not directly imply a more florid manifestation of the disorder. This could be due to the fact that routine testing of a single tissue, such as blood, may not actually reflect the level of UPD in the tissues most susceptible to overgrowth in BWS [START_REF] Smith | Severe presentation of Beckwith-Wiedemann syndrome associated with high levels of constitutional paternal uniparental disomy for chromosome 11p15[END_REF] .

Comparison of mitotic and meiotic recombination breakpoints at 11p

Postfertilization errors such as nondisjunction with reduplication, mitotic recombination, or gene conversion might lead to complete or partial isodisomy.

Mosaicism might be frequent with postfertilization errors, since there would be no selection against the original disomic line. The site of breakpoints were different in all patients suggesting that in contrast to some common breakpoints observed in meiosis there seem to be no common mitotic recombination regions [START_REF] Cooper | Mitotic recombination and uniparental disomy in Beckwith-Wiedemann syndrome[END_REF][START_REF] Russo | Molecular and genomic characterisation of cryptic chromosomal alterations leading to paternal duplication of the 11p15.5 Beckwith-Wiedemann region[END_REF] (Table II).

Correlation of the tests

We found that a combination of two tests (MS-MLPA + MS-HRMA or MS-MLPA + pyrosequencing) is the most useful approach for clinical diagnosis. SNP-arrays alone has been useful for diagnosing UPD11p, however it cannot discriminate between paternal and maternal contribution. The costs of the studies are also important since the SNP-arrays are still expensive and should be set up in experienced laboratories. The limitations of our study are that only 9 patients were evaluated and only one tissue (blood) has been tested in these patients. In contrast, we applied five different techniques to approach patUPD11p, including high density SNP-arrays, which in fact it would have been useful itself to solve most of the issues because it can interrogate not only the dosage but also can demonstrate the percentage of mosaicism and its extent through the short arm of chromosome 11.

Summing up, SNP-arrays have been useful and very informative for clinical diagnosis in patients with BWS with mosaic UPD11p and might be used as the unique tool in clinical diagnostic laboratories, not only to evaluate mosaicism but also to map the mitotic site and consequently the extent of UPD at the short arm of chromosome 11.

Table I .

 I Estimated percentage of cells with UPD calculated for each STR markers (a); methylation index calculated by MS-MLPA (b), MS-HRM (c) and pyrosequencing (d) for each patient. For the methylation analysis it has been calculated the normal methylation index as controls' mean value and standard deviation, for each technique.

		a
				STR markers
	Patient	D11S4046	D11S1318	D11S4088	D11S1338	D11S1346	TH01
	UPD 1	85,77%	82,01%	77,77%	53,49%	-	89,38%
	UPD 2	16,82%	47,91%	31,82%	48,47%	50,52%	37,52%
	UPD 3	-	57,44%	21,49%	-	51,23%	41,39%
	UPD 4	74,14%	73,08%	73,62%	51,26%	67,87%	64,08%
	UPD 5	33,35%	45,58%	40,59%	-	74,83%	63,51%
	UPD 6	81,70%	66,48%	31,26%	-	9,07%	36,31%
	UPD 7	12,93%	28,35%	32,39%	22,79%	-	-
	UPD 8	50,19%	-	63,11%	50,46%	53,73%	49,17%
	UPD 9	58,78%	38,82%	73,84%	37,68%	45,54%	-
		b		c		d
		MS-MLPA	MS-HRM	CpG Q-Pyro
	Patient	H19DMR 1	KvDMR	H19DMR	KvDMR	H19DMR	KvDMR
	UPD 1	114,77%	8,89%	60,98%	24,19%	90,61%	10,96%
	UPD 2	81,25%	31,70%	46,01%	48,91%	73,15%	27,94%
	UPD 3	59,81%	42,76%	46,17%	53,24%	64,80%	28,31%
	UPD 4	79,99%	21,38%	50,41%	40,36%	86,78%	20,50%
	UPD 5	73,47%	23,22%	50,84%	36,46%	79,22%	18,59%
	UPD 6	69,27%	33,98%	51,99%	50,68%	72,07%	26,05%
	UPD 7	70,32%	42,05%	44,37%	48,52%	67,75%	29,13%
	UPD 8	69,09%	34,47%	52,41%	45,96%	74,26%	28,34%
	UPD 9	86,18%	35,85%	51,89%	50,48%	73,26%	22,99%
	Controls' mean	50,26%	54,69%	39,64%	59,67%	52,11%	40,68%
	Std. Desv.	7,18%	4,63%	2,07%	3,74%	2,02%	5,17%

Table II .

 II SNP-array analyses results. The minimum and maximum size estimated for the UPD extent and the gap between these two values are shown. The breakpoints were estimated by means of the identification of the last SNP affected by UPD and the first SNP in heterozygosis. The lower and higher B allele frequencies for all SNPs were calculated. Taking into account that isodisomy is paternal, the percentage of mosaicism of UPD cells is considered to be represented by the frequency of superior B allele.

	Patient	Latest UPD SNP	Position	First Het SNP	Position	Min UPD Size (Mbp)	Gap (Kbp)	Max UPD Size (Mbp)	Inferior B Allele Freq	Superior B Allele Freq	Stand dev
	UPD 1	rs10838427	45,138,258	rs11038507 45,495,455 45,138	357,197	45,495	10,5%	89,5%	4,8%
	UPD 2	rs10838591	46,230,719	rs8914	46,655,700 46,231	424,981	46,656	31,6%	68,4%	4,2%
	UPD 3	rs35834377	41,774,946	rs11036703 42,105,122 41,775	330,176	42,105	35,5%	64,5%	4,2%
	UPD 4	rs11038116	44,680,447	rs704664	44,743,777 44,681	63,330	44,744	23,1%	76,9%	4,6%
	UPD 5	rs1061022	32,083,259	rs7105777	32,997,914 32,083	914,655	32,998	23,1%	76,9%	4,3%
	UPD 6	rs2063082	6,624,904	rs11040978 6,690,629	6,625	65,725	6,691	35,7%	64,3%	6,5%
	UPD 7	rs10832514	2,645,102	rs151288	2,742,878	2,645	97,776	2,743	39,1%	60,9%	3,3%
	UPD 8	rs2177482	31,758,933	rs11031505 31,855,115 31,759	96,182	31,855	30,0%	70,0%	3,5%
	UPD 9	rs2288249	46,290,815	rs876701	46,327,343 46,291	36,528	46,327	29,4%	70,6%	3,7%

Titles and legends to figures

Supplementary Table I.

Spearman's rank correlation coefficient (or Spearman's rho) is a non-parametric measure of statistical dependence between two variables. It assesses how well the relationship between two variables can be described using a monotonic function. In the ovals the correlation coefficients and the statistical significant of the correlation are highlighted. In the vertical ovals there is no correlation between the size of UPD fragment, the percentage of methylation at both imprinting centres and the level of mosaicism. In the horizontal oval are shown a direct correlation between the level of mosaicism and the percentage of methylation of H19DMR (r=+0.933) and an inverse correlation (r=-0.833) between the level of mosaicism and the percentage of methylation of KvDMR; both correlations are statistically significant. II

Supplementary Table