847 ORIGINAL ARTICLE J Med Genet: first published as 10.1136/jmg.2005.032367 on 6 May 2005. Downloaded from Global analysis of uniparental disomy using high density genotyping arrays S Bruce, R Leinonen, C M Lindgren, K Kivinen, K Dahlman-Wright, M Lipsanen-Nyman, K Hannula-Jouppi, J Kere ............................................................................................................................... J Med Genet 2005;42:847–851. doi: 10.1136/jmg.2005.032367 See end of article for authors’ affiliations ....................... Background: Uniparental disomy (UPD), the inheritance of both copies of a chromosome from a single parent, has been identified as the cause for congenital disorders such as Silver-Russell, Prader-Willi, and Correspondence to: Dr Ju Kere, Department of Angelman syndromes. Detection of UPD has largely been performed through labour intensive screening of Biosciences at Novum, DNA from patients and their parents, using microsatellite markers. CBT, 7th Floor, Karolinska Methods: We applied high density single nucleotide polymorphism (SNP) microarrays to diagnose whole Institutet, S-14157 chromosome and segmental UPD and to study the occurrence of continuous or interspersed heterodisomic Huddinge, Sweden; juha. [email protected] and isodisomic regions in six patients with Silver-Russell syndrome patients who had maternal UPD for chromosome 7 (matUPD7). Received24February2005 Results: We have devised a new high precision and high-throughput computational method to confirm Revised form received UPD and to localise segments where transitions of UPD status occur. Our method reliably confirmed and 25 April 2005 Accepted for publication mapped the matUPD7 regions in all patients in our study. 5 May 2005 Conclusion: Our results suggest that high density SNP arrays can be reliably used for rapid and efficient ....................... diagnosis of both segmental and whole chromosome UPD across the entire genome. niparental disomy (UPD) occurs when a child receives Detection of UPD has largely been performed through both copies of a particular chromosome (or part of a labour intensive screening with microsatellite markers of chromosome) from only one parent, thus distorting the DNA from patients and their parents.12 Other means of U 1 fundamental concept of biparental inheritance. UPD may identifying UPD are methylation specific PCR or bisulphite involve two copies of the same chromosome (isodisomy) or sequencing of known imprinted genes on the chromosome of one copy of the contributing parent’s pair of chromosomes interest.13 14 In this study, we set out to explore the new (heterodisomy). Meiotic recombination events can result in a generation of hybridisation based single nucleotide poly- mixed UPD with interspersed regions of heterodisomy and morphism (SNP) genotyping methods, such as the isodisomy along the chromosome. UPD can result from the Affymetrix GeneChip human mapping 10K array http://jmg.bmj.com/ fertilisation of aneuploid gametes, with either gametic (HMA10K), as a simple tool for studying UPD.15 We present complementation or trisomic rescue as the mechanism. A statistical means for diagnosing UPD, including a new non-disjunction event occurring during meiosis I or II is multipoint mapping method that can be used to identify likely to be the original error in these instances. In addition, isodisomic and heterodisomic regions on the autosomal somatic events such as mitotic recombination (causing chromosomes of the genome. The regions are identified by segmental UPDs) or duplication of a viable chromosome to calculating binomial probabilities with 95% confidence compensate for an inherited dysfunctional chromosome can intervals (CI) against genomic background, under the 23 also result in UPD. Imprinting is defined as the bias in the assumption that only parts of the genotyped regions are on September 29, 2021 by guest. Protected copyright. expression of certain genes depending on the parent of origin, affected by UPD. We specifically studied previously diagnosed and is generally associated with parent specific methylation SRS patients with matUPD7 and one patient with suspected patterns of DNA established in the germline.4 If imprinted matUPD7, and found that segments of isodisomy and genes reside on the UPD chromosome, distortion of their heterodisomy can be mapped with great precision and expression patterns will occur and may cause specific certainty. We also provide further evidence for the involve- phenotypes or syndromes,3 as is the case with patUPD11 ment of imprinted genes in the SRS phenotype of matUPD7 and Beckwith-Wiedemann syndrome (MIM #130650), patients. matUPD15 and Prader-Willi syndrome (MIM #176270), and patUPD15 and Angelman syndrome (MIM #105830).56 MATERIALS AND METHODS Furthermore, recessive, normally non-penetrant alleles in Study subjects isodisomic regions may cause recessive diseases.7 Patients and/or parents provided written informed consent, Maternal UPD of chromosome 7 (matUPD7) is found in after which blood samples were obtained. The study was around 10% of patients with Silver-Russell syndrome (SRS; approved by the ethics review board of the Hospital for MIM #180860), suggesting that imprinted genes play a role Children and Adolescents, University of Helsinki, Finland. in its aetiology.8 SRS is a congenital syndrome manifested by Six parent–offspring trios were included in the study, of intrauterine and postnatal growth retardation and typical which five had previously been reported to have matUPD7 dysmorphic features.8 A segmental matUPD reported for and one was a suspected case based on analysis of 12 7q31-qter and cytogenetic abnormalities for the 7p11-p14 chromosome 7 microsatellite markers, which had shown region found in SRS patients delineate two potential regions paternal alleles missing for eight markers (patient designated for imprinted candidate genes for SRS.910 A recessive allele explaining the association between matUPD7 and SRS is Abbreviations: HMA10K, human mapping 10K array; SNP, single unlikely, as a common isodisomic region in matUPD7s has nucleotide polymorphism; SRS, Silver-Russell syndrome; UPD, not been found.11 uniparental disomy www.jmedgenet.com 848 Bruce, Leinonen, Lindgren, et al matUPD7 VII) (table 1).916 This patient was born after and heterodisomy was defined as long stretches of identical J Med Genet: first published as 10.1136/jmg.2005.032367 on 6 May 2005. Downloaded from induced labour for intrauterine growth retardation at 34 genotypes gh between child and parent. Only markers with a gestational weeks to a 30 year old mother and 31 year old successful genotype for all individuals of the trio were father of average height. Her birth length was 39 cm included in the analysis. In our test, the trial was successful if (23.7 SD) and weight 1425 g (23.1 SD). During the an SNP had the genotype gh or gi with the constant genomic pregnancy, slightly elevated alpha fetoprotein levels were probabilities of success fh or fi, respectively. We required all followed up by amniocentesis, which gave a normal female trials in the heterodisomic and isodisomic regions to be karyotype. Otherwise, the pregnancy was uneventful. The successful and the binomial probabilities child has two siblings with normal growth and development. At 4 months of age, the patient was suspected to have SRS and because of her severe growth retardation (25.2 SD) and n n typical dysmorphic features, including leanness (weight in could be expressed simply as fh and fi . P values were n n proportion to height 216%), relative macrocephaly (head Bonferroni corrected by dividing fh and fi by the total circumference 22 SD), prominent ears, a large fontanelle, number of putatively isodisomic and heterodisomic regions, and clinodactyly. As she grew older, she continued to show respectively. The 95% confidence interval was defined by slight dysmorphic SRS-like features, and similar speech and excluding c/2 SNPs from both ends of the UPD regions, where c c feeding difficulties to the other matUPD7 patients in this c satisfies the condition fh ,0.05|max(c) and fi ,0.05|- study.916In addition, she had delayed bone age and lumbar max(c), respectively (that is, a two tailed p value of ,0.05). scoliosis, but did not show classical SRS features such as We used the statistics software R for binomial probability asymmetry or hemihypertrophy, a triangular face, micro- calculation and for the creation of the graphs.18 gnathia, or downturned mouth corners. Her motor and neuropsychological development was slightly delayed and she had premature puberty at 9 years of age. She has normal Power estimation growth hormone and thyroid functions. Her height pro- We estimated the power of our method by calculating the ceeded at 25 SD throughout her childhood and at her last minimum number of SNPs for the UPD regions to be detected evaluation at 12 years of age, her height was 23.0 SD. using HMA10K with 95% power at p = 0.05. Firstly, we calculated the minimum number of mendelian errors (e)in chromosomes without uniparental disomy, for the cumula- Genotyping tive binomial probability of observing at least e mendelian We used the Affymetrix Mapping10K_Xba142 assay, which errors to be ,0.05. This was performed using the mendelian enables the simultaneous genotyping of over 10,000 SNPs using error frequency fn calculated from the number of mendelian only 250 ng of genomic DNA (Affymetrix Inc., Santa Clara, CA, errors in chromosomes 1–6 and 8–22 in the matUPD7 I, II, III, 15 USA). The genotyping was carried