Brigitte Hw Faas
email: b.faas@antrg.umcn.nl

Ineke Van Der Burgt

Angelique Ja Kooper

Rolph Pfundt

Jayne Y Hehir-Kwa

Arie Pt Smits

Nicole De Leeuw

IDENTIFICATION OF CLINICALLY SIGNIFICANT, SUBMICROSCOPIC CHROMOSOME ALTERATIONS AND UPD IN FOETUSES WITH ULTRASOUND ANOMALIES USING GENOME WIDE 250K SNP ARRAY ANALYSIS

Keywords: prenatal, ultrasound, genome wide, SNP array

Background:

The implementation of microarray analysis in prenatal diagnostics is a topic of discussion, as rare CNVs with unknown/uncertain clinical consequences are likely to be found. The application of targeted microarrays limits such findings, but the potential disadvantage is that relevant, so far unknown, aberrations might be overlooked. Therefore, we explore the possibilities for the prenatal application of the genome wide 250k SNP array platform.

Methods: Affymetrix 250k NspI SNP array analysis was performed on DNA from 38 prenatally karyotyped foetuses with ultrasound anomalies. Analyses were performed after TOP, IUFD, or birth on DNA isolated from foetal or neonatal material.

Results: Aberrations were detected in 17/38 foetuses, six of whom with a previously identified chromosomal abnormality and eleven with previously normal or balanced karyotypes. Of the latter, the detected aberration occurred de novo and was considered of clinical relevance in five cases (16%), inherited from a healthy parent in four cases (12%), and de novo yet with unclear clinical relevance in two cases (6%).

The clinically relevant abnormalities were either novel copy number variants (CNVs) (N=3) or concerned a uniparental disomy (UPD; N=2).

Conclusion:

In at least 16% of foetuses with ultrasound anomalies and a normal or balanced karyotype, causal (submicroscopic) aberrations were detected, illustrating the importance of the (careful) implementation of microarray analysis in prenatal diagnosis. The fact that the identified, clinically relevant, aberrations would have gone undetected with most targeted approaches underscores the added value of a genome wide approach.

INTRODUCTION

Chromosome abnormalities have been reported in 9 to 27% of pregnancies in which (major) ultrasound anomalies have been observed. [START_REF] Dallaire | Prenatal diagnosis of fetal anomalies during the second trimester of pregnancy: their characterization and delineation of defects in pregnancies at risk[END_REF][START_REF] Benn | Changes in the utilization of prenatal diagnosis[END_REF][START_REF] Tseng | Detection of chromosome aberrations in the second trimester using genetic amniocentesis: experience during 1995-2004[END_REF] Although limited by its relatively low resolution, conventional cytogenetic analysis using G-or R-banded metaphase spreads still remains the gold standard in prenatal cytogenetics. As a consequence, aberrations smaller than 5-10 Mb may remain undetected. Microarray analysis, a widely accepted technique in postnatal diagnosis, allows for a genome wide scan of unbalanced genomic aberrations at a higher resolution without the need to culture cells. The majority of pregnancies with ultrasound anomalies show a normal karyotype and are therefore likely to benefit from high resolution analysis.

However, as reviewed by Friedman [START_REF] Friedman | High-resolution array genomic hybridization in prenatal diagnosis[END_REF] and Kuehn [5], the use of microarrays in prenatal diagnosis remains an ongoing debate, in particular due to the possible detection of Copy Number Variants (CNVs) for which the clinical consequences are uncertain or unknown. To avoid such findings, most papers so far report on the use of targeted or relatively low-resolution arrays, either during or after pregnancy. [START_REF] Caignec | Detection of genomic imbalances by array based comparative genomic hybridization in fetuses with multiple malformations[END_REF][START_REF] Sahoo | Prenatal diagnosis of chromosomal abnormalities using array-based comparative genomic hybridization[END_REF][START_REF] Shaffer | Comparison of micro-array-based detection rates for cytogenetic abnormalities in prenatal and neonatal specimens[END_REF][START_REF] Van Den Veyver | Clinical use of array competitive genomic hybridization (aCGH) for prenatal diagnosis in 300 cases[END_REF][START_REF] Bi | Rapid prenatal diagnosis using uncultured amniocyted and oligonucleotide array CGH[END_REF][START_REF] Vialard | Array Comparative Genomic hybridization in prenatal diagnosis: another experience[END_REF][START_REF] Rickman | Prenatal detection of unbalanced chromosomal rearrangements by array CHG[END_REF][START_REF] Kleeman | Use of array comparative genomic hybridization for prenatal diagnosis of fetuses with sonographic anomalies and normal metaphase karyotype[END_REF] The application of such targeted arrays has the advantage of being able to carefully select the genomic regions under study, with the ability to focus on regions with known clinical relevance. However, as Tyreman et al. [START_REF] Tyreman | High resolution array analysis: diagnosing pregnancies with abnormal ultrasound findings[END_REF] recently stated, the obvious disadvantage of using a targeted approach is that yet unknown clinical important aberrations might be missed. Moreover, to be kept up-to-date, targeted array platforms need to be redesigned every time a new relevant locus is discovered.

For postnatal analysis genome wide screening with a high resolution array is now general practice and the application of the same platform in prenatal diagnosis would, from a laboratory point of view, be more practical.

In our laboratory, the Affymetrix 250k SNP array is routinely used for genome wide postnatal analysis. We studied the applicability of this array platform in prenatal diagnosis for pregnancies with referral reason "ultrasound anomalies, highly suspect for a chromosomal aberration". Bearing in mind that its high resolution might give rise to results with unknown significance, the analyses were not performed during pregnancy, but only after Termination of Pregnancy (TOP), Intra Uterine Foetal Death (IUFD) or birth. Although in the setting of this study there was no limited time frame, in clinical prenatal practice this usually is the case. Therefore, DNA isolated from uncultured amniocytes was mostly used, to test the suitability of this DNA on our platform.

MATERIALS AND METHODS

Samples

SNP array analyses were performed on DNA from 38 foetuses or newborns who, during pregnancy, showed significant structural malformations with or without intrauterine growth restriction (defined as an estimated fetal weight below the 5th percentile for gestational age), mostly observed at routine ultrasound examination, offered to all pregnant women in The Netherlands around 20 weeks of gestation.

In 36 cases, routine prenatal chromosome analysis was performed following standard procedures as part of the routine diagnostic work-up. Subsequently, after birth, TOP or IUFD, array analysis was diagnostically carried out since, despite having normal or balanced karyotypes, the foetus / child remained suspect for a chromosomal aberration (N = 31: foetuses 1-27, 29-32), or to further characterize a previously identified chromosomal aberration (N = 5: foetuses A-D and F). In two cases (cases E and 28), culturing of the foetal material could not be carried out and therefore only array analysis was performed.

Table I gives an overview of the clinical aspects of the foetuses / newborns.

DNA

The starting foetal / newborn material that was used for DNA isolation is mentioned in Table I. All foetal DNA isolations were performed using the QIAamp DNA Mini Kit (QIAgen, Westburg bv, The Netherlands), following the instructions of the manufacturer. For DNA isolation from uncultured amniocytes, 3-4 ml of amniotic fluid was used and the isolated DNA was subsequently eluted in 50 µl of elution buffer.

DNA from uncultured blood cells from parental blood was isolated following standard procedures (Chemagic Magnetic Separation Module 1 from Chemagen).

Affymetrix NspI SNP array hybridisation and analysis

All analyses were carried out on the Affymetrix GeneChip 250k (NspI) SNP array platform (Affymetrix, Inc., Santa Clara, CA), which contains 25-mer oligonucleotides representing a total of 262,264 SNPs. Hybridisations were performed according to the manufacturer's protocols, with quality criteria of SNP call >85% (based on the dynamic model algorithm GTYPE) and a standard deviation <0.20. Copy numbers were determined using CNAG version 2.0 (Copy Number Analyzer for Affymetrix GeneChip mapping) software package [START_REF] Nannya | A robust algorithm for copy number detection using high-density oligonucleotide single nucleotide polymorphism genotyping arrays[END_REF], by comparing SNP intensities from patient DNA with those of a sex-matched pooled reference DNA sample (DNA from either ten healthy male or ten healthy female individuals). The normalized 2 log ratios were subsequently analyzed for genomic imbalances by a standard Hidden Markov Model (HMM) [17]. Losses were considered significant if at least five consecutive SNP probes showed a value of ≤-0.38 and gains if at least seven consecutive SNPs showed a value of ≥0.3 [START_REF] Hehir-Kwa | Genome-wide copy number profiling on high-density bacterial artificial chromosomes, single-nucleotide polymorphisms, and oligonucleotide microarrays: a platform comparison based on statistical power analysis[END_REF]. CNVs called by the HMM were compared with publicly available and in-house databases (essentially as described in Koolen et al. [START_REF] Koolen | Genomic microarrays in mental retardation: a practical workflow for diagnostic applications[END_REF]) and excluded from further analysis if the observed gain or loss was reported three times or more in the Database of Genomic Variants (DGV: http://projects.tcag.ca/variation/) and/or twice or more in our own database of more than 800 healthy individuals [19; Vrijenhoek et al., Homozygous deletions in the human genome; what don't we need?, Submitted]. Gains or losses of <200 kb or <150kb, respectively (patients), or <600 kb and <450 kb, respectively (parental samples), were only further investigated if genes / gene-rich regions were involved, based on the 95% power analysis described by Hehir-Kwa et al.. [START_REF] Hehir-Kwa | Genome-wide copy number profiling on high-density bacterial artificial chromosomes, single-nucleotide polymorphisms, and oligonucleotide microarrays: a platform comparison based on statistical power analysis[END_REF] The breakpoint positions of each aberrant region were remapped to UCSC hg18 (UCSC Genome Browser, release March 2006) using hgLiftOver (http://genome.ucsc.edu/cgi-bin/hgLiftOver).

In addition to copy number analysis, SNP array data analysis also enables genotyping of the array data and the detection of homozygous stretches.

RESULTS

All array experiments resulted in data that met our quality criteria. The SNP calls from arrays with DNA from uncultured amniocytes versus cultured cells were respectively: 10 vs 12% for SNP calls between 85 and 90%, 52 vs 41% for calls between 90 and 95% and 38 vs 47% SNP calls > 95%. In 15 samples CNVs were detected. Furthermore, in three foetuses large stretches of homozygosity were identified, in two due to uniparental disomy, in the third (case 26) due to consanguinity of the parents. (For overview of all results: see Table I).

Copy number variants

In 15 cases, one or more potentially clinically significant CNV was detected (see Table I; in each of the cases A, F and 27 two CNVs were detected). These CNVs can be categorized into one of the groups of foetuses and CNVs listed below:

1. CNVs detected in foetuses with previously identified chromosomal aberrations

(N=5: foetuses A-D, F)
The analyses of DNA from foetuses in this category were performed to further characterize previously identified pathogenic chromosomal aberrations.

In foetus A, intrauterine growth retardation and pericardial effusion were noticed upon ultrasound examination at 27.4 weeks of gestation. Chromosome analysis on amniocytes revealed a recombinant chromosome 4, resulting from a paternal inv(4)(p16.?3q27). The pregnancy was terminated upon this finding. Further mapping of the breakpoints resulted in 46,XX,rec [START_REF] Friedman | High-resolution array genomic hybridization in prenatal diagnosis[END_REF] Postnatally, the child remained suspect for a 22q11.2 deletion and MLPA analysis using the SALSA MLPA kit P250 was carried out according to instructions of the manufacturer (MRC Holland, Amsterdam, the Netherlands). With this kit, a 22q11.21 deletion was detected that did not encompass the HIRA gene and overlapped with the distal part of the commonly deleted 3 Mb region in DiGeorge syndrome. [START_REF] Demczuk | DiGeorge syndrome and related syndromes associated with 22q11.2 deletions. A review[END_REF] Array analysis resulted in a 46,XX.arr snp 22q11.21q11.22 [START_REF] Mcmullan | Molecular karyotyping of patients with unexplained mental retardation by SNP arrays: a multicenter study[END_REF]390,690,650) In foetus D, with prenatal karyotype 46,XX,del(5)(q35) dn, ultrasound examination at 24.5 weeks of gestation showed several anomalies (see Table I). The child was born at 36.1 weeks of gestation. Axial hypotonia was present from birth. Because of central apnoea and respiratory insufficiency, she was admitted to the neonatal intensive care unit and array analysis was requested for accurate determination of the deleted region. This resulted in a 46,XX,del(5)(q35) dn.arr snp 5q35.2q35.3(175,503,757-180,629,495)x1 karyotype.

In foetus F severe ultrasound anomalies such as cardiac defect and renal anomalies were noticed upon ultrasound examination. Karyotype analysis revealed a 46,XX,del(8)(p23) karyotype and the parents decided to terminate the pregnancy. Subsequent array analysis not only showed the expected loss of 8p, but also a gain of 9p (arr snp 8p23.2p22(180,568-18,676,556)x1,9p24.3p23(30,910-9,903,373)x3).

Karyotype and FISH analysis (subtelomeric regions of 8p and 9p) of both parents showed this unbalanced translocation to be de novo.

2. CNVs detected in foetuses in which no routine cytogenetic analysis could be performed (N=2: foetuses E and 28)

The parents of foetus E, in whom severe ultrasound malformations were observed, were each carrier of a balanced translocation. As the couple had opted for assisted reproduction using a donor egg, the unbalanced karyotype of the father was expected. However, with karyotype arr snp 4p16.3q12(19,099-56,358,811)x3 (56.5

Mb gain), the foetus appeared to have inherited the unbalanced karyotype from the mother, who is a t(4;22)(q12;q11.1) carrier, hinting to an unexpected spontaneous pregnancy. No aberrations on chromosome 22 or elsewhere in the foetal genome were observed.

Only a limited amount of amniotic fluid was submitted for foetus 28 because of IUFD with unknown cause. Array analysis showed a male profile with an 800 kb maternally inherited loss in Xp21.3 (see Figure 1D), a region encompassing three RefSeq genes: MAGEB6, MAGEB18, and VENTXP1.

3. CNVs detected in foetuses with a normal or balanced prenatal karyotype but suspect of a chromosomal aberration (N=8: foetuses 19, 20, 22, 23, 24, 26, 27 and 30) a. Rare, de novo and pathogenic CNVs (foetuses 22, 23, and 24) Foetus 22 showed several ultrasound anomalies (see Table 1) based on which the pregnancy was terminated. Array analysis showed a loss in 11q14.1q14.2 that was originally only detected by visual inspection of the array plots and not by significantly lowered HMM values (see Figure 1A: mean 2 log ratio -019). As this profile was suspect for a mosaic deletion 11q14, metaphase FISH analysis was performed with probes located within the aberrant region of 11q (BAC probes RP11-118L16 and RP11-157B22). This indeed revealed an 11q14.1q14.2 deletion in ~50% of the cells, the other cells showing a normal pattern. As the starting material was DNA isolated from cultured chorionic villi and the foetus was a female, QF-PCR (Aneufast, Genomed, UK) was performed (according to the instructions of the manufacturer) to rule out maternal contamination. No 1B).

Foetus 24 showed a polyhydramnios and a possible oesophagus atresia upon ultrasound examination and died unexpectedly intrauterine. Array analysis showed a 2.9 Mb loss in 17p13.2p13.1 (46,XY.arr snp 17p13.2p13.1(4,421,993-6,972,362)x1 dn) (see Figure 1C).

b. Rare, de novo and suspect CNVs (foetus 30)

In foetus 30 (see Table I) a novel and de novo 640 kb gain in 7p21.2 was found, encompassing (part of) the DGKB gene (karyotype 46,XY.arr snp 7p21.2(14,352,157-14,991,857)x3 dn: see Figure 1F). The DGKB gene product belongs to the family of diacylglycerol kinases (DGKs), regulators of the intracellular concentration of the second messenger diacylglycerol (DAG), which play a key role in cellular processes. In foetus 26 (for ultrasound anomalies: see Table I), array analysis showed a 150 kb gain in 16q23.2 (79,755,648-79,904,890), encompassing two RefSeq genes, PKD1L2 and BCMO1. Moreover, a high percentage of homozygosity (~79 Mb), due to consanguinity of the parents, was observed genome wide in the array profile of this foetus, especially at chromosome 17 (~31 Mb). Parental analysis not only showed the father of the foetus to be carrier of the same gain in 16q23.2, but also revealed an unexpected finding in the (healthy) mother, as she carried a 610 kb loss in 3p25.1p24.3 (arr snp 3p25.1p24.3(15,953,709-16,538,531)x1). The loss comprises four RefSeq genes that have not previously been described to be involved in any pathogenesis. As the mother is a healthy woman, this loss is therefore probably without clinical significance.

Because of a Dandy Walker malformation, the parents of foetus 27 decided to terminate their pregnancy. Upon array analysis, two CNVs were found, an 890 kb paternally inherited gain in 1q42.3q43 and a 400 kb maternally inherited gain in 9p24. [START_REF] Tseng | Detection of chromosome aberrations in the second trimester using genetic amniocentesis: experience during 1995-2004[END_REF]

Uniparental disomy

In two cases, SNP array analysis revealed a single, large homozygous stretch.

In foetus 21 (see Table 1) was prematurely born at 35.6 weeks gestational age by caesarean section. Postnatally, the child remained suspect for a chromosomal anomaly (see Table 1). As can be seen in Figure 1F, SNP array analysis showed a 9.5 Mb homozygous region at the q-terminal part of chromosome 16. Subsequent array analysis of parental samples enabled patient-parent trio analysis and revealed that the child received both chromosomes 16 from his mother, the 9.5 Mb isodisomic region being generated by recombination during meiosis I.

Foetus 29 prenatally showed renal anomalies and polyhydramnios.

Postnatally, the boy showed several anomalies (see Table 1) and array analysis revealed homozygosity of chromosome 4 (see Figure 1G). Patient-parent trio analysis revealed a paternal (iso)uniparental disomy.

DISCUSSION

In the present study, we have evaluated the application of the Affymetrix whole genome 250k SNP array, which is routinely used in our laboratory for postnatal diagnostic analyses in patients with mental retardation and / or congenital anomalies.

In our series of 38 samples, six cases were subjected to array analysis for further characterization of a previously identified aberration or, because both parents were translocation carrier and karyotyping was not possible (cases A-F). In all these cases no interpretational dilemmas were experienced and all detected CNVs were classified as clinically significant.

Thirty-two other foetuses (cases 1-32) were analysed because ultrasound anomalies were detected and yet a normal or balanced foetal karyotype was found (N=31), or because insufficient vital cells were available for routine cytogenetic analysis (N=1). In this group, 11 foetal DNA samples revealed CNVs that could not be detected by routine chromosome analysis (11/32 = 34%). Five of these could be classified as clinically relevant (5/32 = 16%), two as with unclear clinical significance (2/32 = 6%) and four (4/32 = 12%) were inherited.

In 3/5 cases with clinically relevant CNVs (cases 22, 23 and 24), the de novo losses were several Mb in length, encompassing tens of genes (~10-50). In all these cases none of the genes were known to be directly linked to known diseases that could account for the ultrasound anomalies. In the two other foetuses uniparental disomies of chromosome 16 (maternal) and 4 (paternal) were observed. As maternal UPD [START_REF] Hehir-Kwa | Genome-wide copy number profiling on high-density bacterial artificial chromosomes, single-nucleotide polymorphisms, and oligonucleotide microarrays: a platform comparison based on statistical power analysis[END_REF] has been reported in literature [START_REF] Kotzot | Uniparental disomy (UPD) other than 15: phenotypes and bibliography updated[END_REF] and ECARUCA (www.ecaruca.net) to cause growth retardation, renal agenesis and heart defects, this finding is considered as most likely causative for the clinical features of the boy. Pathological consequences of paternal UPD(4) have not been described so far. [START_REF] Kotzot | Uniparental disomy (UPD) other than 15: phenotypes and bibliography updated[END_REF] However, we consider it highly likely that this finding in foetus 29 is clinically relevant since, as a result of the UPD, a recessive gene mutation may be unmasked. The possible presence of a low percentage of mosaicism in the foetus can also not be excluded, although this is not very likely as only a rescue from a monosomy 4 can explain the complete iso-uniparental profile. The results of the array and amniotic fluid analysis do not suggest a mosaic profile in these tissues.

In two other foetuses, aberrations were detected of which, even after parental analysis, the clinical relevance remains unclear. In case 28, the clinical relevance of the inherited X-chromosomal loss encompassing three genes, that have not previously been reported to be involved in any pathogenesis, is difficult to determine.

Although copy variants in the MAGE and VENXP1 genes are not likely to cause pathogenesis, a clinical relevance cannot be ruled out with certainty without further family investigation.

In the mother, the loss might be compensated by the remaining X chromosomal allele, possibly in combination with skewed X-inactivation, compensatory processes that are obviously absent in the male foetus. The 7p21.2 gain in foetus 30 has not previously been reported and as the DGKB gene is not known to be involved in pathogenic processes, the clinical relevance of this gain, although suspect, remains unclear.

Our results illustrate the additional value and diagnostic yield of the use of genome wide SNP array analysis in prenatal diagnosis in pregnancies with ultrasound anomalies and a normal or balanced karyotype. Since most of the clinically significant aberrations had not previously been recognized as pathogenic CNVs, they would have gone undetected with most of the targeted array approaches described by others. [START_REF] Caignec | Detection of genomic imbalances by array based comparative genomic hybridization in fetuses with multiple malformations[END_REF][START_REF] Sahoo | Prenatal diagnosis of chromosomal abnormalities using array-based comparative genomic hybridization[END_REF][START_REF] Shaffer | Comparison of micro-array-based detection rates for cytogenetic abnormalities in prenatal and neonatal specimens[END_REF][START_REF] Van Den Veyver | Clinical use of array competitive genomic hybridization (aCGH) for prenatal diagnosis in 300 cases[END_REF][START_REF] Bi | Rapid prenatal diagnosis using uncultured amniocyted and oligonucleotide array CGH[END_REF][START_REF] Vialard | Array Comparative Genomic hybridization in prenatal diagnosis: another experience[END_REF][START_REF] Rickman | Prenatal detection of unbalanced chromosomal rearrangements by array CHG[END_REF][START_REF] Kleeman | Use of array comparative genomic hybridization for prenatal diagnosis of fetuses with sonographic anomalies and normal metaphase karyotype[END_REF] Nonetheless, whole genome screening can, even with the inclusion of parental analysis, also lead to the detection of CNVs with unknown or uncertain clinical relevance, as is illustrated by the CNVs in cases 28 and 30. Whole genome parental analysis can also lead to the detection of new, unclear CNVs in healthy parents, as illustrated for case 26. We, however, argue that these disadvantages do not outweigh the great diagnostic power of whole genome SNP array analysis, as clearly illustrated by the high diagnostic yield is this study (16% clinical significant aberrations vs 6% with unknown clinical relevance).

Our results also illustrate the importance of the availability of parental DNA, in order to be able to distinguish between a rare, inherited variant and a de novo, (most likely) pathogenic imbalance, as in 4/11 foetuses with novel CNVs these could be classified as inherited from a healthy parent. Although rare inherited CNVs cannot always with certainty be classified as without clinical relevance, they are, dependent on the size and type of CNV, less likely to directly lead to a clinical phenotype: small CNVs (<0.1 Mb) and gains are less likely to be pathogenic than large CNVs (>1 Mb) and losses, respectively. [23,24] We agree with Friedman [START_REF] Friedman | High-resolution array genomic hybridization in prenatal diagnosis[END_REF] and Coppinger [25] that in a prenatal setting, it is not only highly recommended to be able to include parental analysis upon the finding of a suspicious CNV, but also to be able to obtain the parental samples simultaneously with the foetal sample. This because of the limited time frame prenatal diagnosis is dealing with but obviously also to avoid as much as possible that parents will receive results with unknown clinical significance.

Previously, Tyreman et al. [START_REF] Tyreman | High resolution array analysis: diagnosing pregnancies with abnormal ultrasound findings[END_REF] studied the possible prenatal use of the high resolution Affymetrix GeneChip 6.0 SNP array and retrospectively studied 106 anonymized cultured samples of foetuses that were prenatally karotyped because of major ultrasound anomalies or multiple soft markers. In 9% of the foetuses they detected likely pathogenic CNVs, in 11% likely benign CNVs and in 12% CNVs of unknown clinical significance. But, as the study was performed with anonymized samples, parental analysis to obtain more information on the nature of the CNVs could not be carried out. As Tyreman et al. used detection criteria comparable to ours, the difference between the results (9 vs 16% likely pathogenic) can probably be explained by the difference in numbers of samples that were included and the inclusion of parental samples in our study.

Shaffer et al. [START_REF] Shaffer | Comparison of micro-array-based detection rates for cytogenetic abnormalities in prenatal and neonatal specimens[END_REF] and Kleeman et al. [START_REF] Kleeman | Use of array comparative genomic hybridization for prenatal diagnosis of fetuses with sonographic anomalies and normal metaphase karyotype[END_REF] both studied the use of targeted aCGH using Signature microarrays and in their cohorts of patients only 1.3% and 2%, respectively, showed a chromosome abnormality that was considered causal for the phenotype. Van den Veyver et al. [START_REF] Van Den Veyver | Clinical use of array competitive genomic hybridization (aCGH) for prenatal diagnosis in 300 cases[END_REF] tested DNA isolated from 84 foetuses with ultrasound anomalies during pregnancy, on targeted BAC arrays and in three of these foetuses (4%) CNVs of uncertain clinical significance that would have remained undetected with karyotyping were found. These relatively low percentages of abnormal findings can probably be explained by the use of a targeted array instead of high resolution whole genome arrays, as most of the clinically relevant abnormalities detected with our platform would not have been detected with the platforms used by these groups. Indeed, in a recently published comparative study using aCGH, Coppinger et al.

[25] also concluded that whole-genome array analysis is preferred over targeted array analysis.

The group of Bi [START_REF] Bi | Rapid prenatal diagnosis using uncultured amniocyted and oligonucleotide array CGH[END_REF] also used targeted arrays for analysis of foetal material during pregnancy. In their study they showed the feasibility of prenatal genetic diagnosis using oligonucleotide arrayCGH analysis for direct analysis of amniocytes without culturing cells. Since short handling times are an extremely important issue in prenatal diagnosis, especially as ultrasound anomalies are often only detected around 20 weeks of gestation, when structural ultrasound scans are routinely being carried out, we also tested the use of DNA isolated from uncultured amniotic fluid cells on the 250K SNP array platform. In all cases where this type of DNA was available (see Table I), array results that met our quality criteria could be obtained.

In conclusion, genome wide SNP array analysis offers a highly reliable way to accurately detect the highest number of clinically relevant imbalances and homozygous findings compared to any other (set of) test(s) in human clinical genetics. Therefore, despite potential results with unknown or uncertain clinical relevance, this technique is ready for implementation in daily practice of prenatal diagnosis for pregnancies highly suspect for chromosomal aberrations. Of course results with uncertain or unknown clinical significance are from a counselling and patient point of view undesired, but they are inherent to testing, regardless of the technique being used. The maximum effort possible should be undertaken to avoid unclear results and for genome wide array analysis in a prenatal setting, this could be achieved by adjusting both counselling and analytical and reporting approaches, as compared to the postnatal setting. [17] Criteria for losses and gains (such as size and number of probes) might be chosen differently from those used in a postnatal setting.

One might agree to only report results with known clinical consequences in case of very small aberrations and screen genome wide for larger imbalances, based on constantly increasing knowledge in publicly available and in-house databases. If this is carefully surveyed by extensive pre-and posttest counselling by skilled clinical geneticists and array analysis can be performed within a limited time frame, we are convinced that the time is right for implementation. Table I: Overview of the clinical aspects of the foetuses / newborns included in the study, the pregnancy history, the foetal / newborn material that was used for DNA isolation, array results and its clinical relevance.

  c. Rare, inherited and most likely without clinical relevance CNVs (foetuses 19, 20, 26, and 27) Foetus 19 showed cerebral abnormalities upon ultrasound examination. SNP array analysis revealed a 50 kb loss in 7p21.3, that involved an intronic region of the NXPH1 gene. Parental array analysis showed the mother of the foetus to be carrier of the same loss, the final karyotype being 46,XX.arr snp 7p21.3(8,476,409-8,526,074)x1 mat. Foetus 20 showed exencephalus upon ultrasound examination. SNP array analysis showed a 340 kb paternally inherited gain in 17q12 (foetal karyotype: 46,XY.arr snp 17q12(31,159,050-31,496,575)x3 pat).

  diagnosis of constitutional disorders. Nat Genet 2007;39:S48-S54. 24 Vermeesch JR, Fiegler H, de Leeuw N, Szuhai K, Schoumans J, Ciccone R, Speleman F, Rauch A, Clayton-Smith J, Van Ravenswaaij C, Sanlaville D, Patsalis PC, Firth H, Devriendt K, Zuffardi O. Guidelines for molecular karyotyping in constitutional genetic diagnosis. Eur J Hum Genet 2007;15:1105-1114. 25 Coppinger J, Alliman S, Lamb AN, Torchia BS, Bejjani BA, Shaffer LG. Wholegenome microarray analysis in prenatal specimens identifies clinically significant chromosome alterations without increase in results of unclear significance compared to targeted microarray. Prenat Diagn 2009;29:1156-1166.

  Termination of Pregnancy; IUFD = Intrauterine Foetal Death ** UC = Uncultured; U = Cultured; AF = Amniotic Fluid; CHOR = Chorionic villi; FIBRO = fibroblasts Legend to Figure 1: Chromosome copy number variation and homozygous regions identified by high resolution SNP array analysis in patients 21 (A), 22 (B), 23 (C), 24 (D), 28 (E), 29 (F) and 30 (G). The log 2 T/R (test-over-reference) ratio values are plotted on the Y-axis versus the genomic position on the respective chromosome represented by the ideogram on the X-axis in the lower part of the figure. The red dots in the upper panel represent individual SNP values. The thin blue line in this panel represents the effective HMM outcome with a normal (N=2) T/R ratio of 0. A significant imbalance is indicated by a rise or fall of this line by 0.3 identifying a single copy number gain and -0.38 identifying a single copy number loss. In the lower panel (blue) each dot represents the averaged value of 10 neighbouring SNPs. An idiogram of the chromosome involved is shown on the bottom. A blue line below the ideogram reflects regions of homozygosity, not being caused by a loss. The regions of interest are denoted by black circles. A: Case 22. A 3.6 Mb loss in 11q14.1q14.2 is visible. Note the small decrease in that region, suggesting a mosaic loss. B: Case 23. A 5 Mb loss in 3q26.2q21.3. C: Case 24. A 2.9 Mb loss in 17p13.2p13.1. D: Case 28. A 800 kb loss in Xp21.3. The blue line at the bottom of the figure indicates homozygosity of the X-chromosome, consistent with a male profile. E: Case 30. A 640 kb gain in 7p21.2. F: Case 21. The thin blue line at the bottom of the figure shows a 9.5 Mb homozygous region on chromosome 16. G: Case 29: The blue line at the bottom of the figure shows homozygosity of chromosome 4.

F

  upd(16)mat.arr snp 1-22(256.553 SNPs)x2,X(5.710 SNPs)x1 A arr snp 11q14.1q14.2(82,843,560-86,402,152)x1~2 dn B arr snp 3q26.33q27.2(181,214,043-186,173,910)x1 C arr snp 17p13.2p13.1(4,421,993-6,972,362)x1 dn D arr snp Xp21.3(25,724,049-26,543,804)x0 mat G arr snp 4p16.2q35.2(19,099-191,167,888)hmz E arr snp 7p21.2(14,352,157-14,991,857)x3 dn

Figure 1 :

 1 Figure 1: SNP array profiles of aberrant cases

  maternal contamination was detected. Parental analysis showed normal profiles and the final karyotype was 46,XX.arr snp 11q14.1q14.2(82,843,560-86,402,152)x1~2 dn (3.6 Mb loss (mosaic)).

	The mother of foetus 23 underwent amniocentesis at 16.2 weeks of gestation
	because of advanced maternal age, and a 46,XX,t(3;18)(q26.2;q21.3)dn karyotype
	was found. At 20 weeks gestational age, the foetus developed a hydrocephalus and
	the pregnancy was terminated. Array analysis on cultured amniotic fluid cells
	revealed a 5 Mb loss in the long arm of chromosome 3 at the breakpoint of the
	translocation: 46,XX,t(3;18)(q26.2;q21.3)dn.arr snp 3q26.33q27.2(181,214,043-
	186,173,910)x1 (see Figure

  (foetal karyotype: 46,XY.arr snp 1q42.3q43(233,888,438-234,778,604)x3 pat,9p24.3(619,468-1,022,153)x3 mat).

ACKNOWLEDGEMENTS

The authors want to thank the technicians of the Array and the Prenatal and Postnatal Diagnostics Groups of the Department of Human Genetics, Radboud University Nijmegen Medical Centre, for technical support, clinical geneticists prof dr HG Brunner, prof dr N van Slobbe -Knoers, dr BA Koolen, dr T Kleefstra from the Department of Human Genetics, Radboud University Nijmegen Medical Centre, and dr R Oegema from the Erasmus University Medical Centre Rotterdam, for referring patients, the various gynaecologists from the Network for Prenatal Diagnosis Nijmegen, The Netherlands for their kind cooperation, and dr M Swinkels from the Division of Human Genetics of the University Medical Centre of Utrecht, The Netherlands, for kindly providing information on case B.

COPYRIGHT LICENCE STATEMENT

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in Journal of Medical Genetics and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://group.bmj.com/products/journals/instructions-for-authors/licenceforms).

COMPETING INTEREST

None declared.


R S Van Der Post

L A Kiemeney

M J L Ligtenberg

J A Witjes

C A Hulsbergen-Van De Kaa

D Bodmer

L Schaap

C M Kets

J H J M Van Krieken

MD Nicoline Hoogerbrugge
email: n.hoogerbrugge@antrg.umcn.nl

The Risk of Urothelial Bladder Cancer in Lynch syndrome is increased, in particular among MSH2 mutation carriers

Keywords: Lynch Syndrome, HNPCC, urothelial cancer, bladder cancer, MSI Lynch Syndrome, HNPCC, urothelial cancer, bladder cancer, MSI

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

Introduction

Lynch syndrome, previously called Hereditary Non-Polyposis Colon Carcinoma (HNPCC), is caused by a germline mutation in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2. It results in a large increase in spontaneous mutations and thus has a direct oncogenic effect. Besides the high risk of developing colorectal carcinomas of 10 to 80%, Lynch syndrome family members are at increased risk of developing several extra-colonic cancers and tumours at a relatively young age: endometrial cancer, carcinomas of the ovary, small bowel and biliary tract cancer, sebaceous gland tumours and urothelial carcinomas (UC) of the upper urinary tract. [START_REF] Aarnio | Life-time risk of different cancers in hereditary non-polyposis colorectal cancer (HNPCC) syndrome[END_REF][START_REF] Vasen | MSH2 mutation carriers are at higher risk of cancer than MLH1 mutation carriers: a study of hereditary nonpolyposis colorectal cancer families[END_REF][START_REF] Dunlop | Cancer risk associated with germline DNA mismatch repair gene mutations[END_REF][START_REF] Hendriks | Cancer risk in hereditary nonpolyposis colorectal cancer due to MSH6 mutations: impact on counseling and surveillance[END_REF][START_REF] Quehenberger | Risk of colorectal and endometrial cancer for carriers of mutations of the hMLH1 and hMSH2 gene: correction for ascertainment[END_REF][START_REF] Sijmons | Urinary tract cancer and hereditary nonpolyposis colorectal cancer: risks and screening options[END_REF][START_REF] Lynch | Multiple primary cancer, including transitional cell carcinoma of the upper uroepithelial tract in a multigeneration HNPCC family: molecular genetic, diagnostic, and management implications[END_REF][START_REF] Vasen | Cancer risk in families with hereditary nonpolyposis colorectal cancer diagnosed by mutation analysis[END_REF][START_REF] Watson | Extracolonic cancer in hereditary nonpolyposis colorectal cancer[END_REF][START_REF] Baglietto | Risks of Lynch Syndrome Cancers for MSH6 Mutation Carriers[END_REF] The lifetime risk of upper urinary tract cancer in Lynch syndrome varies in different studies from 0.4% to 20%. [START_REF] Aarnio | Life-time risk of different cancers in hereditary non-polyposis colorectal cancer (HNPCC) syndrome[END_REF][START_REF] Vasen | MSH2 mutation carriers are at higher risk of cancer than MLH1 mutation carriers: a study of hereditary nonpolyposis colorectal cancer families[END_REF][START_REF] Sijmons | Urinary tract cancer and hereditary nonpolyposis colorectal cancer: risks and screening options[END_REF][START_REF] Watson | Extracolonic cancer in hereditary nonpolyposis colorectal cancer[END_REF][START_REF] Vasen | Guidelines for the clinical management of Lynch syndrome (hereditary non-polyposis cancer)[END_REF][START_REF] Watson | The risk of extra-colonic, extra-endometrial cancer in the Lynch syndrome[END_REF][START_REF] Aarnio | Cancer risk in mutation carriers of DNA-mismatch-repair genes[END_REF][START_REF] Maul | Extracolonic cancers associated with hereditary nonpolyposis colorectal cancer in the Utah Population Database[END_REF][START_REF] Parc | Cancer risk in 348 French MSH2 or MLH1 gene carriers[END_REF][START_REF] Geary | Generelated cancer spectrum in families with hereditary non-polyposis colorectal cancer (HNPCC)[END_REF][START_REF] Lin | Cumulative incidence of colorectal and extracolonic cancers in MLH1 and MSH2 mutation carriers of hereditary nonpolyposis colorectal cancer[END_REF][START_REF] Bermejo | Cancer characteristics in Swedish families fulfilling criteria for hereditary nonpolyposis colorectal cancer[END_REF] MSI is present in these urothelial carcinomas of the upper urinary tract. [START_REF] Gylling | Differential cancer predisposition in Lynch syndrome: insights from molecular analysis of brain and urinary tract tumors[END_REF][START_REF] Hartmann | Frequent microsatellite instability in sporadic tumors of the upper urinary tract[END_REF] A study based on the Swedish family cancer database, quantifying the occurrence of UC in families with at least 4 generations showed that families fulfilling the Bethesda criteria [START_REF] Umar | Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability[END_REF] have an increased risk of cancers in the ureter, but not in the urinary bladder. [START_REF] Bermejo | Cancer characteristics in Swedish families fulfilling criteria for hereditary nonpolyposis colorectal cancer[END_REF] It was concluded that in families at risk for Lynch syndrome, UC of the bladder occurs at a frequency comparable to that in the general population. The study did not report results which were stratified by type of mutation. On the contrary, in a study by Geary et al, bladder cancer was more common in MSH2 mutation families than expected in the general population, the relative risk (RR) being 3.6, P=0.001. [START_REF] Geary | Generelated cancer spectrum in families with hereditary non-polyposis colorectal cancer (HNPCC)[END_REF] Cumulative risks were not presented. In their study every case of bladder cancer was accompanied by cancer of the ureter in the family. For that reason, the authors concluded that the risk of bladder cancer was increased only in families with ureter cancer.

In addition to morphologic resemblance, sporadic urothelial cell cancers of the renal pelvis, ureter and bladder share the most important risk factors and molecular genetic aberrations; it is therefore remarkable that the risk of bladder cancer does not appear to have increased in Lynch syndrome families, while the risk of upper urinary tract cancer has. The aim of the present study was to establish whether patients with Lynch syndrome are at increased risk of cancer of the urinary bladder.

Material and methods

Patient population

From 95 Lynch syndrome families, we selected all carriers of a germline mutation in the MSH2, MLH1 or MSH6 gene, who had been seen and registered at the Department of Human Genetics of the Radboud University Nijmegen Medical Centre between 1996 and November 2008, and all their first-degree relatives: a total of 627 men and 617 women. Information on mutation carriers and their first-degree relatives was systematically collected by postal questionnaires and telephone calls with contact persons when data were outdated for more than 1 year. In each family, one to six such persons were contacted in order to obtain up-to-date information on the occurrence of carcinoma in mutation carriers and their first-degree relatives. Firstdegree relatives with MMR-gene mutation negative tests were excluded. All diagnoses of UC carcinoma were verified and confirmed by review of pathology reports or medical reports.

Statistical analysis

We calculated follow-up time for each family member with the date of birth as starting point until the dates of last contact, death, or diagnosis of cancer, whichever came first. Kaplan-Meier (KM) survival analyses were used to calculate the cumulative risk (plus standard error) of cancer until specific ages. We chose the age of 70 as censuring age because the number of patients being at risk after that age is small. SPSS version 16.0 was used for KM analyses. For reference, we used life-table cancer risk estimates as reported by the Dutch Cancer Registry (www.ikcnet.nl), based on cancer incidence and population demographics in 2003. [START_REF] Kiemeney | The risk of cancer in the Netherlands[END_REF] The life-table risks of urinary tract cancer are almost identical to those reported by SEER for whites in the U.S.A. (seer.cancer.gov). Relative risks of cancer among specific groups compared to the Dutch reference risks were defined as the ratio between observed and expected numbers of tumours before the age of 70 years. Exact, 95% confidence intervals of the relative risks were calculated using the publicly available OpenEpi software (www.openepi.com).

Histology and molecular analysis

After written informed consent, histology reports and tissue blocks of UC of the bladder, ureter or renal pelvis were collected. Clinicopathological features of the urothelial cell cancers were reviewed by two experienced pathologists using haematoxylin and eosin (H&E) slides. The following parameters were scored: 1) tumour differentiation according to the WHO 2004 grading system; 2) T-stage (TNM 2002); 3) Growth pattern: papillary or solid; 4) presence of tumour-infiltrating lymphocytes (TILs): absent, moderate or dense.

For microsatellite instability (MSI) analysis, areas of the formalin fixed and paraffin embedded tissues containing either tumour cells (at least 30%) or normal cells were marked and material from these areas was isolated separately using a lysis buffer and a protein precipitation solution (Purogene, Gentra systems, Minneapolis). PCR of the markers D2S123, D5S346, D17S250, BAT25, BAT26 and BAT40 was performed using standard conditions in the presence of a fluorescently labelled primer.

GeneScan analysis was performed on an ABI373A, ABI3100 or ABI3700 apparatus (PE Biosystems, Foster City, CA). A tumour was considered MSI positive, when at least 2 of these standard set of markers, or 30% of an extended set, showed instability. [START_REF] Kets | Very low prevalence of germline MSH6 mutations in hereditary nonpolyposis colorectal cancer suspected patients with colorectal cancer without microsatellite instability[END_REF] Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tissues. Slides were stained with antibodies against MLH1 (Pharmingen code: 51-1327gr), PMS2 (Pharmingen code: 556415), MSH2 (Oncogene Research Products code: NA26) and MSH6 (Transduction Laboratories code: G70220). Staining patterns of MMR proteins were evaluated using normal epithelial, stromal and inflammatory cells as internal controls. Stained slides were scored as (1) positive, i.e. showing nuclear staining in at least some tumour cells; (2) negative, i.e. no staining of the tumour with a positive internal control or (3) not assessable, i.e. insufficient technical quality to provide an unambiguous result despite repeated assays. [START_REF] De Jong | Microsatellite instability, immunohistochemistry, and additional PMS2 staining in suspected hereditary nonpolyposis colorectal cancer[END_REF][START_REF] Overbeek | Interpretation of immunohistochemistry for mismatch repair proteins is only reliable in a specialized setting[END_REF] As a verification 5 anonymous sporadic cancers of the bladder were taken at random from the files of the Department of Pathology.

Results

Members of 95 Lynch syndrome families diagnosed by a germline mutation in MLH1

(n=26), MSH2 (n=43) or MSH6 (n=26) were questioned by contact persons for occurrence of cancer such as UC of the bladder, ureter or renal pelvis. The total cohort of 1244 persons consisted of 406 proven mutation carriers (individuals with a pathogenic mutation on germline mutation analysis) and 838 first-degree relatives.

The latter group consisted of 111 obligate mutation carriers (concluded because of their position in the pedigree in relation to relatives tested positive for a mutation) and 727 persons at 50% risk to be mutation carriers. In 22 families, at least one (n=27) confirmed mutation carrier or first-degree relative of a mutation carrier was diagnosed with UC of the urinary bladder, ureter or renal pelvis. In two additional MSH2 families, the patient with UC of the bladder was not a first but a second-degree relative of the mutation carrier diagnosed. Second-degree relatives were not included in the KM risk analysis but the two second degree relatives with UC were included in the histopathology study. UC of the bladder was diagnosed in 21 patients from [START_REF] Gylling | Differential cancer predisposition in Lynch syndrome: insights from molecular analysis of brain and urinary tract tumors[END_REF] Lynch syndrome families, the majority (71%) coming from MSH2 families (15 MSH2, 2 MLH1, and 4 MSH6); 19 out of 21 (90%) were men. Two patients with bladder cancer were at 50% risk of being an MSH2 mutation carrier but could not be tested. Bladder cancer was diagnosed at an average age of 60 ± 12 years (range 41-84), which is approximately 10 years younger than the average age of sporadic bladder cancer in the Netherlands.

The cumulative risk of bladder cancer until the age of 70 (CR70) in all MMR mutation carriers and their first-degree relatives was 7.5% [95% CI 3.1-11.9%] for men and 1.0% [95% CI 0-2.4%] for women. (Table 1) The corresponding CR70 for the Dutch population is 1.8% for men and 0.5% for women. The relative risk for carriers of any MMR mutation, as compared to the general Dutch population, was 4.2 [95% CI 2.2-7.2, p<0.001] for men and 2.2 [95% CI 0.3-8.0, p=0.5] for women (table 1). MSH2 mutation carriers and first-degree relatives showed the highest risk of bladder cancer: CR70 is 12.3% [95% CI 4.3-20.3%] in men and 2.6% [95% CI 0-3.9%] for women.

The relative risk of bladder cancer in carriers of an MSH2 mutation and their firstdegree relatives, as compared to the general Dutch population, was 7.0 [95% CI 3.4-13.0, p<0.001] for men and 5.8 [95% CI 0.5-16.1, p=0.15] for women. The overall CR70 risk for urinary tract cancer, including the bladder, in MSH2 mutation carriers and first-degree relatives was 18.2% [95% CI 5.0-31.4%] in men and 8.4% [95% CI 0-15.4%] in women (table 1).

Two or more primary urinary tract cancers of which one in the bladder were diagnosed in 9 of all 21 patients with bladder cancer; in six patients these two cancers were diagnosed synchronously or within 1 year. The remaining 3 patients, with a metachronous combination of bladder and upper urinary tract cancer, had their bladder cancer diagnosed earlier in life than the upper urinary tract cancer. Exclusive upper urinary tract cancer was diagnosed in 8 patients from [START_REF] Lynch | Multiple primary cancer, including transitional cell carcinoma of the upper uroepithelial tract in a multigeneration HNPCC family: molecular genetic, diagnostic, and management implications[END_REF]

Discussion

This study indicates an increased risk of urothelial cancer of both the urinary bladder and the upper urinary tract (ureter and renal pelvis) in patients with Lynch syndrome carrying a germline MSH2 mutation. Furthermore, this study indicates that cancer of the urinary bladder, ureter and renal pelvis is also, though rarely associated with MSH6 or MLH1 mutations. A causal relation between MSH2 deficiency and bladder cancer is likely. In the first place because bladder cancers in these families often show MSI and/or absence of IHC staining of the MSH2 protein, just like upper urothelial tract cancer and colorectal cancer. In the second place because of the presence of two MSH2 mutation families, each of which contained two first-degree family members with bladder cancer, and with three of the bladder cancers diagnosed under the age of 50 (as illustrated in figure 3). It is interesting that especially MSH2 mutation carriers are at increased risk of urothelial cancer, as observed in this cohort and in previous studies. [START_REF] Vasen | MSH2 mutation carriers are at higher risk of cancer than MLH1 mutation carriers: a study of hereditary nonpolyposis colorectal cancer families[END_REF][START_REF] Watson | The risk of extra-colonic, extra-endometrial cancer in the Lynch syndrome[END_REF][START_REF] Geary | Generelated cancer spectrum in families with hereditary non-polyposis colorectal cancer (HNPCC)[END_REF] The diversity in the function of the MSH2, MLH1 and MSH6 protein might be responsible for the variation in cancer risk. Environmental factors (e.g. smoking status) may also affect the urologic tract cancer occurrence rates but we do not have the necessary information to determine if this might contribute to the differences. In 8 out of 21 patients with bladder cancer, this was their first cancer diagnosis whereas at this stage 5 of them developed another Lynch syndrome associated cancer at an older age. Therefore, early diagnosis of Lynch syndrome may prevent development of a second primary cancer, especially CRC by regular colonoscopy with polypectomy. [START_REF] Jarvinen | Controlled 15-year trial on screening for colorectal cancer in families with hereditary nonpolyposis colorectal cancer[END_REF] The diagnosis of Lynch syndrome in healthy relatives may lead to prevention or early detection of cancer, which improves the prognosis. [START_REF] Jarvinen | Controlled 15-year trial on screening for colorectal cancer in families with hereditary nonpolyposis colorectal cancer[END_REF] Thus bladder cancer, just like upper urothelial tract cancer, can be added to the Bethesda criteria [START_REF] Umar | Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability[END_REF] and the Amsterdam II criteria [START_REF] Vasen | New clinical criteria for hereditary nonpolyposis colorectal cancer (HNPCC, Lynch syndrome) proposed by the International Collaborative group on HNPCC[END_REF] and lead to the clinical suspicion of Lynch syndrome. The occurrence of bladder cancer can be used in family history taking and molecular diagnostics to identify the families that are at risk of Lynch syndrome.

Cumulative risks of colorectal and endometrial cancer are within the ranges published by other studies. [2-5, 8, 13, 28, 29] Bladder cancer risk observed in our cohort was significantly higher than that observed in previous studies. [START_REF] Sijmons | Urinary tract cancer and hereditary nonpolyposis colorectal cancer: risks and screening options[END_REF][START_REF] Watson | Extracolonic cancer in hereditary nonpolyposis colorectal cancer[END_REF][START_REF] Watson | The risk of extra-colonic, extra-endometrial cancer in the Lynch syndrome[END_REF][START_REF] Geary | Generelated cancer spectrum in families with hereditary non-polyposis colorectal cancer (HNPCC)[END_REF][START_REF] Bermejo | Cancer characteristics in Swedish families fulfilling criteria for hereditary nonpolyposis colorectal cancer[END_REF] These studies differed from our study by a) type of risk that was calculated, b) by type of population and c) whether or not the type of mutation was distinguished. The data of these studies are given in Table 3. Additionally the discrepancy in bladder cancer risk between our study and other studies may result from our systematic data collection approach (obtaining of up-to-date information). This led to the discovery of nine new cases of bladder carcinoma, previously unrevealed with the standard procedure of a clinical geneticist taking the family history.

Considering the high risk of urothelial tract cancer in MSH2 mutation carriers, a surveillance program needs to be developed. At present various recommendations have been published. The present European guidelines for families with a MMR mutation include ultrasound and urine analysis every 1-2 years with patients from the age of 30 to 35 only in cases where upper urinary tract cancer runs in the family (two or more cases of UC). [START_REF] Vasen | Guidelines for the clinical management of Lynch syndrome (hereditary non-polyposis cancer)[END_REF] The American guidelines include urinalysis with cytology every 1-2 years with patients from the age of 25 to 35 for all family members with Lynch syndrome. [START_REF] Lindor | Recommendations for the care of individuals with an inherited predisposition to Lynch syndrome: a systematic review[END_REF] Watson et al proposed (unspecified) surveillance of patients starting with the age of 50, especially for families that carry mutations in MSH2. [START_REF] Watson | The risk of extra-colonic, extra-endometrial cancer in the Lynch syndrome[END_REF] Although cytology is the superior marker in terms of specificity, [START_REF] Lokeshwar | Bladder tumor markers beyond cytology: International Consensus Panel on bladder tumor markers[END_REF] Myrhoy et al showed that the sensitivity of urine cytology in diagnosing asymptomatic upper urinary tract cancer in Lynch syndrome is approximately 30%. Therefore, cytology only is not a proper method of surveillance. [START_REF] Myrhoj | Screening for urinary tract cancer with urine cytology in Lynch syndrome and familial colorectal cancer[END_REF] The most important biomarker of urothelial cancer is macro-or microscopically haematuria, which occurs in 85% of patients with bladder cancer. [START_REF] Wakui | Urinary tract cancer screening through analysis of urinary red blood cell volume distribution[END_REF] Consequently, Koornstra et al recommend annual surveillance for haematuria by urinary dipstick, of all patients with Lynch syndrome, beginning at the age of 45 to 50. [START_REF] Koornstra | Management of extracolonic tumours in patients with Lynch syndrome[END_REF] The recent use of sensitive transducers has improved imaging of the upper urinary tract and bladder by ultrasonography. It was shown to be as accurate in the detection of renal masses and bladder filling defects as intravenous urography and CT scanning (EAU Guidelines 2008).

Further studies are needed for developing an optimum early detection strategy concerning the appropriate interval, methods to be used and patient groups to be included. Until then, we propose a combination of ultrasonography of the bladder and upper urinary tract, urinary cytology and urine sediment (erythrocytes) every 1 to 2 years. We recommend a surveillance program for UC of the bladder and upper urinary tract for all MSH2 mutation carriers starting at the age of 40, which is based on the youngest Lynch syndrome patient with bladder cancer reported in the literature (age 40) and observed in our study (age 41). [START_REF] Hirata | A novel germline mutation of MSH2 in a hereditary nonpolyposis colorectal cancer patient with liposarcoma[END_REF] The top age limit of surveillance for UC can be similar to that of surveillance for colorectal cancer.

Surveillance should not be limited to families with a history of UC, because in our study clustering of urinary tract cancer was only observed in 5 families, while 19 patients had a negative family history of urinary tract cancer. In conclusion, our data suggest that in addition to upper urinary tract cancer, urothelial cancer of the urinary bladder is part of the Lynch syndrome tumour spectrum. Especially carriers of an MSH2 mutation are at increased risk of urinary tract cancer including cancer to the bladder. In these cases we consider surveillance necessary.

Box 1

 1 Recommendations of UC surveillance in Lynch syndrome 1. Surveillance with a combination of ultrasound of the bladder and upper urinary tract, urinary cytology and sediment 2. In every MSH2 mutation carrier 3. From age 40 and up 4. Performed every 1-2 years

table 2 )

 2 . Three bladder tumours were identified having normal IHC staining and 1 tumour was MSS. As a control, IHC of 5 sporadic bladder carcinomas showed normal staining of all 4 MMR system proteins. No typical histological characteristics were observed in the bladder carcinomas when compared with sporadic bladder carcinomas (table2).

	Lynch syndrome

Acknowledgement

We would like to thank Irene Reimerink and Marsha Voorendt (genetic counselors), Dr Ernie Bongers (clinical geneticist), Riki Willems, Monique Link, Monique Goossens, Sandra Hendriks-Cornelissen and Evelien Hoenselaar (technical assistance).

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in Journal of Medical Genetics and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence.

Competing Interest: None to declare Ethics Committee approval: this is not an intervention study with human subjects; no approval of the Ethics Committee was obtained

Legends to figures


MD Hilde Van Esch
email: hilde.vanesch@med.kuleuven.be

Elisabeth M Rosser

Sandra Janssens

Ingrid Van Ingelghem

Bart Loeys

Björn Menten

Bjorn Menten

Developmental delay and connective tissue disorder in four patients sharing a common microdeletion at 6q13-14

Keywords: chromosome 6q, microdeletion, developmental delay, connective tissue, collagen, COL12A1 Competing Interest: None declared

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

Introduction

A detailed phenotypic description of small interstitial 6q deletions is hampered by the scarcity of such patients and most of the reported cases represent large, cytogenetically detectable deletions [START_REF] Zherebtsov | Further delineation of interstitial chromosome 6 deletion syndrome and review of the literature[END_REF]. The widespread use of array comparative genome hybridisation (array-CGH) in the diagnostic work-up of patients presenting with congenital disorders including developmental delay has enabled the identification of many patients with smaller chromosomal imbalances [START_REF] Buysse | Challenges for CNV interpretation in clinical molecular karyotyping: Lessons learned from a 1001 sample experience[END_REF]. It moreover permitted the identification and delineation of new microdeletion and microduplication syndromes, allowing a more detailed assessment of the phenotypic consequences associated with specific chromosomal imbalances. The present report describes the genotypic and phenotypic delineation of four patients with a de novo interstitial deletion of chromosome 6q13-14, resulting in a common microdeletion of 3.7 Mb. All patients present with developmental delay, mild dysmorphism and signs of lax connective tissue. Interestingly, the COL12A1 gene that maps within the common deleted region constitutes a good candidate for the connective tissue pathology.

Clinical description Patient 1

The girl was born at 35 weeks gestation by spontaneous delivery after an uncomplicated pregnancy to healthy, unrelated parents. Family history is unremarkable. At birth, both feet were in an extreme dorsiflexion position and she had a fibromatosis colli, both necessitating physiotherapy. Initially, feeding was difficult because of general hypotonia with poor sucking. Motor development was delayed because of this hypotonia as well as the presence of hyperlaxity in hips and large joints. She sat with her thighs internally rotated and her knees flexed. She started to crawl at 21 months. In addition, her speech development was delayed with the use of only a few words at the age of two. At her last clinical examination, at the age of 2 years, the growth parameters were all within the normal range. She has mild motor delay and moderate language delay (level of 18 months at age 24 months). She has a high-pitched voice and shows good social contact. Hyperlaxity is still present and she has a pronounced lordosis and bilateral pes plani. She also has mild dysmorphic features including a high forehead, long philtrum with thin upper vermillion, broad neck and sacral dimples (Figure 1A). Brain imaging and EMG were normal, but on radiographs of the spine an anterolisthesis L2-L3 was seen. Because of the hyperlaxity and developmental delay, Larsen syndrome was initially suspected.

Patient 2

This boy was born at 37 weeks of pregnancy. He was hospitalized on the neonatal ward because of low birth weight (2300 g) and repetitive hypoglycemia. He was the first and only child of unrelated healthy parents. Family history revealed borderline mental impairment present in the brother of the father and in his maternal uncle. At the age of 3 years an inguinal unilateral hernia was surgically corrected. His psychomotor development was characterized by walking at 22 months and delayed speech development. He was referred at the age of 5 years because of his delay with an IQ of 50-55.

Clinical examination at that age showed a boy with growth parameters in the high-normal range.

Hyperlaxity was present in fingers, wrists and elbows together with bilateral pes plani with short toes and a mild pectus excavatum. He had mild dysmorphic features including long philtrum with thin upper vermillion, large earlobes, mild synophris and a metopic ridge (Figure 1B). Brain imaging, EEG, echocardiography, radiograph of the spine and metabolic work-up were all normal.

Patient 3

This girl was born at term after an uneventful pregnancy. The neonatal period was complicated by a necrotizing enterocolitis. Because of facial dysmorphism, she was seen by a clinical geneticist at the age of 2 months. Several dysmorphic signs were noted: low frontal hair line, high nasal bridge, long philtrum with thin upper lip, low-set posterior rotated ears, high palate and retrognathia. Her thorax was short and an umbilical hernia was present. She had bilateral tri-phalangeal thumbs and broad distal phalanges. She presented with a bilateral nystagmus, axial hypotonia and hands in a fist position. Re-examination at the age of 5 months showed axial hypotonia as well as hyperlaxity, and length and weight below the third centile. She suffered from recurrent respiratory infections. A bilateral sensorineural hearing loss of 55 dB was documented. Ultrasound of the brain showed a small periventricular hemorrhage. Ultrasound of the abdomen showed bilateral small kidneys.

Radiograph of the skull and spine were normal. She died at the age of 8 months of unknown cause, post-mortem examination was refused by the parents.

Patient 4

This boy is the second child of healthy unrelated parents, with no family history of note. He was born after an unremarkable pregnancy at 38 weeks gestation by planned Caesarian, because of breech position. He was noted to have undescended testes and hypotonia. Chromosome analysis was performed at 20 months of age because of motor delay and dysmorphic features. This showed an apparently balanced translocation, 46,XY t(2:6)(q31;q15). When he was seen in clinic he was noted to have marked joint laxity, a smooth philtrum, thin upper vermillion, upturned nose with anteverted nostrils, large ears, pointed chin and 5 th finger clinodactyly (Figure 1 C,D). At the age of 6 years he was found to have hypogonadotrophic hypogonadism (not Kallman's) and was referred back to the genetics clinic. At that time his parents reported that he was making slow but steady progress. He attended a special school and had some difficulties with fine motor skills. His biggest problem was walking; he had begun to walk at 3 ½ years but still frequently fell due to eversion at the ankles. On examination, his facial features were as described before. He continued to have generalized joint laxity and had mild truncal obesity. His feet appeared small for his size with bilateral pes cavus. He held his feet in eversion with both halluces pointing upwards. A hard swelling was present on the lateral side of each foot which has been ascribed to the fact that his weight bears along the lateral side of the feet. Ophthalmological examination was normal.

Methods

The protocol was approved by the appropriate Institutional Review Board of the University Hospitals of Leuven and Ghent in Belgium, and the Institute of Child Health London, and informed consent was obtained from the parents of the affected patients. Written consent was given to the authors for publication of the patient's images in print and online. Genomic DNA of each patient and the parents was isolated according to standard procedures.

Standard cytogenetic analysis showed normal karyotypes for patients 1, 2 and 3. In patient 4 an apparently balanced de novo translocation, 46,XY,t(2:6)(q31;q15) was detected.

The genomic DNA of patients 1 and 4 was analyzed on the total genome 1Mb BAC array. Clone preparation, hybridization and data analysis were performed as previously described [START_REF] Menten | Emerging patterns of cryptic chromosomal imbalance in patients with idiopathic mental retardation and multiple congenital anomalies: a new series of 140 patients and review of published reports[END_REF]. Both patients 2 and 3 were analyzed using the Agilent Human Genome CGH 44k array [START_REF] Buysse | Challenges for CNV interpretation in clinical molecular karyotyping: Lessons learned from a 1001 sample experience[END_REF]. All genome coordinates mentioned in this study are according to human genome build 18 (NCBI 36.1). The inheritance of each of these deletions was investigated by analyzing parental samples using microarrays (patients 1 and 2) or karyotyping and FISH analysis (patients 3 and 4).

Results

In the absence of an etiological diagnosis in patients 1, 2 and 3, array-CGH analysis was performed.

Regarding patient 4, array analysis was performed to query the balanced nature of the chromosomal rearrangement. Table 1 summarizes the clinical and molecular findings in the 4 patients. All patients carry overlapping deletions of variable size spanning the 6q13-14 region (Figure 1F). The shortest region of overlap (SRO) is defined by the proximal breakpoint at 72.65 Mb in patient 2 and the distal breakpoint at 76.31 Mb in patient 1. This critical region is 3.66 Mb in size and contains 16 annotated genes (Table 2, supplementary). All deletions have arisen de novo.

Discussion

Since the introduction of array-CGH technology, the rate of discovery of novel genomic imbalances has increased dramatically. New disorders are described by their genomic rearrangement and subsequently, clinical features are compared among patients when a common imbalance is identified. In this regard, the use of worldwide databases of chromosomal imbalances such as DECIPHER, has greatly facilitated the identification of additional patients, as in our cases [START_REF] Firth | DECIPHER: Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources[END_REF].

Interstitial deletions involving chromosome 6q13-14 are rare with eight patients reported to date [START_REF] Mcneal | Congenital anomalies including the VATER association in a patient with del(6)q deletion[END_REF][START_REF] Young | Deletions of the long arm of chromosome 6: two new cases and review of the literature[END_REF][START_REF] Gershoni-Baruch | Interstitial deletion (6)q13q15[END_REF][START_REF] Romie | Monosomy 6q1: syndrome delineation[END_REF][START_REF] Hopkin | New insights into the phenotypes of 6q deletions[END_REF][START_REF] Kumar | Proximal interstitial 6q deletion: a recognizable syndrome[END_REF][START_REF] Myers | Proximal 6q interstitial deletion without severe mental retardation[END_REF][START_REF] Lespinasse | Characterization of an interstitial deletion 6q13-q14.1 in a female with mild mental retardation, language delay and minor dysmorphisms[END_REF]. Here, we present four more patients that were analyzed by array-CGH because of developmental delay and facial dysmorphism. By comparing the facial features, we could distinguish some common features, namely long philtrum, thin upper vermillion, pointed chin and large ear lobes (Figure 1). All patients presented with developmental delay with hypotonia, the latter symptom especially in the first years of life. In the older reported patients language and motor delay were noted, with severity correlating with the deletion sizes.

Interestingly, in each patient marked joint laxity was noticed by the respective clinical geneticist, and in patient 1 even the diagnosis of Larsen syndrome was suggested. This hyperlaxity was also noted in some of the older reported patients, with dislocation of the hips in three patients, and flatfeet in two [START_REF] Mcneal | Congenital anomalies including the VATER association in a patient with del(6)q deletion[END_REF][START_REF] Young | Deletions of the long arm of chromosome 6: two new cases and review of the literature[END_REF][START_REF] Gershoni-Baruch | Interstitial deletion (6)q13q15[END_REF][START_REF] Kumar | Proximal interstitial 6q deletion: a recognizable syndrome[END_REF][START_REF] Myers | Proximal 6q interstitial deletion without severe mental retardation[END_REF][START_REF] Lespinasse | Characterization of an interstitial deletion 6q13-q14.1 in a female with mild mental retardation, language delay and minor dysmorphisms[END_REF]. In addition, in the majority of patients, other features of loose connective tissue were present, including umbilical and inguinal hernia and thorax and foot deformities. Within the SRO deletion, the COL12A1 gene is present, which encodes the alpha chain of type XII collagen, a member of the FACIT (fibril-associated collagens with interrupted triple helices) collagen family. Type XII collagen is a homotrimer found in association with type I collagen, an association that is thought to modify the interactions between collagen I fibrils and the surrounding matrix. Expression analysis in zebrafish showed that collagen type XII is broadly expressed in many connective tissues including fascia and basement membranes [START_REF] Bader | Zebrafish collagen XII is present in embryonic connective tissue sheaths (fascia) and basement membranes[END_REF]. Moreover, sequence variants within COL12A1 have recently been associated with increased risk of ligament ruptures in a case-control genetic association study [START_REF] Posthumus | The association between the COL12A1 gene and anterior cruciate ligament ruptures[END_REF]. To date, COL12A1 has not been implicated in human disease, to our knowledge, and no mouse model is available. Our finding might represent the first association between haploinsufficiency of the COL12A1 gene and a possible human phenotype. Further sequencing analysis in a large group of patients with similar connective tissue pathology as well as the development of a mouse model will enable us to confirm these results and open the way to exactly define the phenotype associated with this collagen gene.

Other interesting genes within the common defined deletion are KCNQ5, a voltage dependent potassium channel and member of a very large family of potassium channels of which some have been linked to diseases including cardiac arrhythmias, deafness and epilepsy [START_REF] Jentsch | Neuronal KCNQ potassium channels: physiology and role in disease[END_REF] 2). Mutations in the latter gene cause an autosomal recessive lysosomal free sialic acid storage disease [START_REF] Verheijen | A new gene, encoding an anion transporter, is mutated in sialic acid storage diseases[END_REF]. Lespinasse and co-authors suggested that the 5-HT1B gene could be responsible for the mental retardation and language delay observed in 6q13-14del patients [START_REF] Lespinasse | Characterization of an interstitial deletion 6q13-q14.1 in a female with mild mental retardation, language delay and minor dysmorphisms[END_REF]. This gene encodes a hydroxytryptamine receptor and these type of receptors have been implicated in a variety of neuropsychiatric disorders [START_REF] Sari | Serotonin1B receptors: from protein to physiological function and behaviour[END_REF]. It maps 2 Mb telomeric to the common defined deleted region and is deleted in two of our patients. We can not exclude a role for this gene in the developmental delay of these patients, but given the fact that the two other patients also clearly exhibit a developmental delay, another gene(s) within the SRO should also be involved. In this regard the FILIP gene encoding a Filamine A-interacting protein involved in cortical cell migration represents an interesting candidate [START_REF] Nagano | Filamin A-interacting protein (FILIP) regulates cortical cell migration out of the ventricular zone[END_REF]. Yet, neither of our patients shows neuronal migration defects on MRI scan of the brain.

Another feature present in the majority of patients is urogenital anomalies with cryptorchidism reported in three out of the five male patients. In patient 4, this cryptorchidism is probably due to the documented hypogonadotrophic hypogonadism.

Our analysis of four cases of 6q13-14 microdeletion defines a novel site of copy number variation associated with developmental delay, distinct facial features and connective tissue pathology. The latter feature in particular may constitute a good clinical handle to identify additional patients.

  and SLC17A5 (Table

	Patient	1	2	3	4	Lespinasse et al.[12] Older published
							cases [5-11]
	DECIPHER		00002719	00000863	00000140
	Sex	F	M	F	M	F	3 M/4 F
	size deletion	68213162-76318818 72654054-76925922 72157311-97184863	72157311-80953422	73112000-80988000
	Birth	35 weeks	37 weeks	term	38 weeks	39 weeks
	weight	3005 g	2300 g	2350 g	NI	3780 g
	lenght	47 cm	47.5 cm	47cm	NI	53 cm
	OFC	34 cm	NI	35 cm	NI	37 cm
	Age at first clinical exam	16 months	5 years	2 months	23 months	30 months
	Developmental delay	+	+	+	+	+	7/7
	Motor delay, hypotonia	+	+	+	+	+	7/7
	Mental level	mild delay	IQ = 55	too young to assess	moderate delay	IQ = 70
	Facial dysmorphism						6/7
	thin upper lip	+	+	+	+
	smooth long philtrum	+	+	+	+
	low frontal hair line			+
	high palate	+		+
	retrognathia			+		+
	high nasal bridge	+		+
	low set ears	+	+	+		+
	other				epicanthus, prominent ears epicanthus, hypotelorism
	Growth parameters/age	16 months	5 years	2 months	23 monts	10 years
	weight	10.5 kg (P50)	18.2 kg (P75)	3220 g (< P3)	14.5 kg (P90)	40 kg (>P90)
	lenght	81 cm (P75)	108.5 cm (P50)	50 cm (<P3)	81 cm (P3)	149 cm (>P90)
	OFC	47 cm (P50)	52 cm (P90)	37.4 cm (P25)	49 cm (P10)	54 cm (>P90)
	Joint laxity (all joints)	+ (LL>UL)	+	+	+	+
	thorax		mild pectus	short		pectus excavatum
			excavatum
	loose connective tissue talipes equinovarus	inguinal henia	umbilical hernia	pes cavus	umbilical hernia	5/7
						cubitus valgus
						flat valgus feet
	Urogenital anomalies	VUR grade 2-3		small kidneys	cryptorchidism		3/7
	Other	high pitched voice	sacral dimple	neonatal enterocolitis hypogonadotr. hypogonadism
		sacral dimple		nystagmus	obesity
				died at 8 months
				recurrent respiratory
				infections

Acknowledgements

The authors wish to thank the patients and families for their cooperation. H.V.E. and B.L. are clinical investigators funded by F.W.O. Vlaanderen. We do not have any affiliation with any group with a direct financial interest in the subject matter or materials discussed in the manuscript.

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Legends to the figures

Written consent was given to the authors for publication of the patient's images in print and online. Table 1 Overview of molecular and clinical data of the new and reported patients with a microdeletion 6q13-14. Reported patients are referenced in [START_REF] Mcneal | Congenital anomalies including the VATER association in a patient with del(6)q deletion[END_REF][START_REF] Young | Deletions of the long arm of chromosome 6: two new cases and review of the literature[END_REF][START_REF] Gershoni-Baruch | Interstitial deletion (6)q13q15[END_REF][START_REF] Romie | Monosomy 6q1: syndrome delineation[END_REF][START_REF] Hopkin | New insights into the phenotypes of 6q deletions[END_REF][START_REF] Kumar | Proximal interstitial 6q deletion: a recognizable syndrome[END_REF][START_REF] Myers | Proximal 6q interstitial deletion without severe mental retardation[END_REF][START_REF] Lespinasse | Characterization of an interstitial deletion 6q13-q14.1 in a female with mild mental retardation, language delay and minor dysmorphisms[END_REF]. Abbreviations: F: female; M: male; +: present; NI: no information; LL: lower limbs; UL: upper limbs; VUR: vesico-uretral reflux; hypogonadotr.:

Table 2 Supplementary

List of the reference genes present in the shortest region of deletion overlap at 6q13-q14.1, based on human genome build 18 (NCBI 36.1).