RESEARCH ARTICLE
UPD Detection Using Homozygosity Profiling With a SNP Genotyping Microarray Peter Papenhausen,1* Stuart Schwartz,1 Hiba Risheg,2 Elisabeth Keitges,2 Inder Gadi,1 Rachel D. Burnside,1 Vikram Jaswaney,1 John Pappas,3 Romela Pasion,1 Kenneth Friedman,4 and James Tepperberg1 1
Laboratory Corporation of Cytogenetics Triangle Park, North Carolina
2
Laboratory Corporation of America, Cytogenetics Seattle, Washington
3
NYU School of Medicine, Department of Pediatrics, New York, New York Laboratory Corporation of America, Molecular Genetics, Research Triangle Park, North Carolina
4
Received 30 January 2010; Accepted 6 January 2011
Single nucleotide polymorphism (SNP) based chromosome microarrays provide both a high-density whole genome analysis of copy number and genotype. In the past 21 months we have analyzed over 13,000 samples primarily referred for developmental delay using the Affymetrix SNP/CN 6.0 version array platform. In addition to copy number, we have focused on the relative distribution of allele homozygosity (HZ) throughout the genome to confirm a strong association of uniparental disomy (UPD) with regions of isoallelism found in most confirmed cases of UPD. We sought to determine whether a long contiguous stretch of HZ (LCSH) greater than a threshold value found only in a single chromosome would correlate with UPD of that chromosome. Nine confirmed UPD cases were retrospectively analyzed with the array in the study, each showing the anticipated LCSH with the smallest 13.5 Mb in length. This length is well above the average longest run of HZ in a set of control patients and was then set as the prospective threshold for reporting possible UPD correlation. Ninety-two cases qualified at that threshold, 46 of those had molecular UPD testing and 29 were positive. Including retrospective cases, 16 showed complete HZ across the chromosome, consistent with total isoUPD. The average size LCSH in the 19 cases that were not completely HZ was 46.3 Mb with a range of 13.5–127.8 Mb. Three patients showed only segmental UPD. Both the size and location of the LCSH are relevant to correlation with UPD. Further studies will continue to delineate an optimal threshold for LCSH/UPD correlation. Ó 2011 Wiley-Liss, Inc.
Key words: uniparental disomy; single nucleotide polymorphism; microarray; homozygotic stretch; LOH; copy neutral
How to Cite this Article: Papenhausen P, Schwartz S, Risheg H, Keitges E, Gadi I, Burnside RD, Jaswaney V, Pappas J, Pasion R, Friedman K, Tepperberg J. 2011. UPD Detection Using Homozygosity Profiling With a SNP Genotyping Microarray. Am J Med Genet Part A 155:757–768.
Engel in 1980 while the first proven case of whole chromosome UPD was reported in 1987 [Engel, 1980; Creau-Goldberg et al., 1987]. UPD may be associated with a supernumerary marker chromosome [Robinson et al., 1993], a balanced homologous (isochromosome) or non-homologous Robertsonian translocation [Robinson et al., 1994], or low grade mosaicism/placental confined mosaicism involving the UPD chromosome [Papenhausen et al., 1999]. Those cases involving chromosomes with imprinted genes result in clinical effects dependent on parent of origin. Some cases may be associated with a recessive disorder in which there is only one carrier parent due to isoUPD segments either in a whole UPD chromosome pair or in a partial chromosomal region (segmental isoUPD) secondary to mitotic recombination [Schollen et al., 2005; Reboul et al., 2006]. However, the true incidence of UPD is unknown with many cases likely to arise with few clues, but with distinct clinical risks. Single nucleotide polymorphism (SNP)-based microarrays allow for the designation of regions of base pair homozygosity (HZ) through the targeting of biallelic markers dispersed at high density throughout the genome. Homozygotic markers occur when
INTRODUCTION Uniparental disomy (UPD) is defined as the inheritance of both homologs of a chromosome pair from a single parent. When both homologs of that parent are present, it is referred to as heterodisomy, while isodisomy is the presence of two copies of one parental homolog. The concept of UPD was introduced by Eric
Ó 2011 Wiley-Liss, Inc.
*Correspondence to: Peter Papenhausen, Laboratory Corporation of America 1912 Alexander Dr. Research Triangle Park, NC, 27709 E-mail:
[email protected] Published online 15 March 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ajmg.a.33939
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758 both parents have the same allele at any one target SNP. In some individuals, long contiguous stretches of HZ (>5 Mb) can be observed, and in others shorter, but excessively numerous, runs can be found [Gibson et al., 2006; Li et al., 2006]. The multiple long stretches are known to represent identity by descent (IBD), wherein ‘‘autozygous’’ chromosome segments have been passed from a common ancestor. As these segments segregate and are subdivided by additional generations of recombination, they become fewer and smaller proportional to the degree of outbreeding [Papenhausen et al., 1999; Gibson et al., 2006; McQuillan et al., 2008]. A possible correlate with a long homozygotic stretch in a single chromosome pair is UPD, which is primarily thought to result subsequent to a correction from trisomy or monosomy in the placenta. A review of 36 archival cases of predominately maternal UPD revealed a high (23/36) degree of at least partial isoUPD despite analyses that were often based on a limited number of markers especially those confirming a methylation-based UPD diagnosis. Therefore, it was deemed reasonable that a SNP array would reveal isodisomy related stretches of homozygotic alleles in nearly all UPD cases and that these would extend over many megabases due to limited recombinational events. It was decided to establish a normal curve of the longest single run of HZ from the patient population and to determine a threshold for UPD correlation from retrospectively confirmed UPD cases to use for a prospective pilot study. Our dual objective of this study was to confirm our observations of 15 years of UPD studies which suggested that a UPD ‘‘signature’’ could be provided by SNP microarray analysis without necessitating a parental trio analysis and to attempt to ascertain risks ancillary to UPD.
METHODS All patients in this report were either studied by the Affymetrix SNP microarray retrospective to molecular identification of UPD (9 cases; 8 were in the initial retrospective study and one was added during the study as one of three that followed exclusion of a parental fragile X repeat) or by molecular UPD testing after prospective identification of the suggestive UPD pattern of SNP microarray HZ (29 cases). Nearly all prospective case analyses, including those used to establish a standard distribution of HZ, were patients referred for various symptoms of developmental delay, autism or congenital anomalies. Molecular confirmatory testing of UPD was performed on most samples by standard microsatellite analysis of the parental and patient blood or amniotic fluid cells. One case was confirmed by trio microarray SNP genotype analysis. All positive results required at least two marker parental exclusions in our protocol and were confirmed for paternity. Markers both inside and outside the long contiguous stretch of HZ (LCSH) were included to rule out segmental UPD. Bisulfite methylation pattern confirmation for UPD 11, 14, and 15 was alternatively or additionally performed in cases involving those chromosomes [Kosaki et al., 1997]. Fragile X analysis in three cases was performed by standard PCR and southern blotting techniques [Brown et al., 1993], as well as standard G-band chromosome analysis. The Affymetrix version 6.0 genechip (1.8 million SNP and Copy Number (CN) targets) with GTC 3.1 software was used for most of these studies (The Nsp portion of the 500K genechip was used in
AMERICAN JOURNAL OF MEDICAL GENETICS PART A 10% of the cases with 262,000 SNPs). The >900,000 allele specific SNPs in the 6.0 gene chip provides results used to detect contiguous stretches of HZ (copy neutral loss of heterozygosity) throughout the genome while assessing dosage with the aid of an additional 900,000 non-polymorphic CN probes (Affymetrix 6.0 data sheet). The GTC software setting for display of contiguous HZ was set at >1 Mb with a 1% allowance for outliers. The software provides alleleic differentiation, log 2 dosage, a smoother of the log 2 dosage for estimates of mosaicism, copy number and loss of heterozygosity (LOH). When no heterozygosity is recorded over a 1 Mb stretch, a block appears in the appropriate genomic position. Close analysis of the alleleic differentiation will reveal copy neutral mosaicism found in segmental UPD as tracts diverging from the common midpoint heterozygote 0 line. Copy number estimates are extrapolated from the log 2 dosage.
RESULTS A normal distribution of the longest single stretch of HZ was constructed from SNP microarray patient referral to help establish an optimal threshold for subsequent molecular UPD testing. A consecutive analysis of 120 patients referred for microarray analysis (predominant indication: developmental delay/autism) generated a mean and SD of 3.64 1.7 Mb (Fig. 1). A typical pattern of HZ and allele segregation in chromosome 15 is depicted in Figure 2 and can be compared to molecular and array results of a UPD 15 (case#29, Fig. 3a,b). The initial retrospective analysis of eight confirmed UPD cases resulted in array identification of two cases with total HZ (complete isoUPD) and six displaying the anticipated recombination related LCSH consistent with partial isoUPD with heteroUPD present in the remaining portion(s) of the chromosome. The size of the single LCSH ranged from 13.5 to 48.4 Mb with one case (#27) displaying two LCSH of 11 Mb and 11.2 Mb. (Table I). A threshold for reporting a possible UPD correlation was therefore set at a single chromosomal LCSH 13.5 Mb and an allowance was made for two LCSH in a single chromosome when the sum was >15 Mb. Telomeric LCSH, significant for high linkage disequilibrium (LD), were included when exceeding 5 Mb and the single chromosomal total
FIG. 1. The LCSH distribution of the longest single chromosomal block in 120 consecutive patients studied.
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FIG. 2. Top: HZ blocks from a ‘‘normal’’ biparental chromosome 15. Small blocks of HZ (1–3 Mb) are common in most chromosomes, particularly in patients from restricted population gene pools such as in a small town. Some small blocks at specific loci are common in most populations. Middle: SNP alleles segregate into homozygotes AA, BB in the upper and lower tracts with the heterozygotes in the middle (0 line). Bottom: Copy number state with the chromosome band positions below.
exceeded 15 Mb; these were found only on confimred UPD chromosomes. A LCSH 10 Mb in a second chromosome was considered indicative of IBD and therefore, exclusionary for UPD reporting/follow-up. There were 92 prospective cases that showed the UPD qualifying LCSH and parental follow-up was obtained in 46 cases-mostly involving imprinted chromosomes, of which 29 were confirmed as UPD. Fourteen of the 30 had complete isoUPD, and 13 were a mixture of hetero and isoUPD with a range of LCSH size from 14.9 to 127.8 Mb (Table I). The Affymetrix Genotype Console images of case examples are shown in Fig. 4a–h. Two prospective patients showed telomeric-associated LCSH and biparental markers proximal to the HZ region, consistent with mitotic recombination and segmental UPD. One of these (case 36) showed faint evidence of the excluded parental allele in subsequent molecular analysis of the HZ region (Fig. 5a,b) while the array showed clear evidence of mosaicism for segmental UPD of 11p in a patient with Beckwith–Weidemann syndrome (Fig. 5c). Combining both retrospective and prospective cases, all 19 mixed hetero/isoUPD cases demonstrated a single chromosome with one or more LCSH with a mean total and SD of 46.32 33.57 Mb (range of 13.5–127.8) while 16 patients demonstrated complete isoUPD (Table I). All cases with combined hetero/isodisomy were molecularly confirmed and parent-oforigin established. Thirteen of the 16 completely HZ cases were confirmed, but in three (chromosomes 1, 6, 8) follow-up was not obtained. These cases were considered de facto UPD based on overwhelming LD probability, although parent-of-origin is uncertain. Three retrospective cases, in which fragile X testing revealed trinucleotide repeat sizes that excluded one parent, showed complete isodisomy X in two and segmental UPD for the 30 Mb terminal Xq region in the third, the only retrospective case not part of the
original eight. The first of these was a female referred for infertility, who exhibited a single paternal FMR1 premutation and no maternal allele. The microarray revealed complete X chromosome HZ; additional microsatellite testing showed maternal exclusion and paternal isoallelism consistent with paternal isoUPD X. The molecular testing in a second fragile X related case (Fig. 4g) showed only the known maternal full mutation in an XXY male (Klinefelter syndrome). Microsatellite analysis showed multiple paternal exclusions and a single maternal allele while the SNP microarray revealed complete X chromosome HZ consistent with maternal isoUPD X. The third case involved the follow-up of a prenatal 46,XX karyotype with a molecular result showing only the maternal full mutation and no paternal allele. Microsatellite analysis revealed paternal exclusions closely flanking the FMR1 gene and biparental markers in the short arm to Xq21. The microarray analysis showed a copy neutral 30 Mb LCSH from Xq25 to the Xq telomere. Microsatellite confirmation and LCSH in one of the three segmental UPD cases (#36) is shown in Figure 5a,b. The allele ratio segregation in the segmental UPD BWS case (#37) is shown in Figure 5c. Sixteen cases with qualifying LCSH were found not to be associated with UPD upon molecular follow-up. These patients are shown in Table II. Common patterns of UPD associated LCSH are shown in Figure 6a. Examples of regions with qualifying LCSH not associated with UPD are shown in Figure 6b. In two of these regions, centromeric 11 and 13q21, fraternal brothers were seen with a matching, and thus biparentally inherited LCSH that would otherwise have qualified for follow-up. None of the false positives had qualifying telomeric LCSH. The same distribution of LCSH in non-UPD chromosomes of the hetero-iso UPD patients was found as in the controls. One patient with a 61.9 LCSH in the UPD group had two 9 Mb runs in
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FIG. 3. a: Microsatellite analysis confirming paternal exclusions associated with maternal UPD 15 and Prader-Willi syndrome in case #29. Two of the marker systems (boxes) were informative showing paternal exclusions. The combination of isodisomy and heterodisomy depend on meiotic recombination sites and were seen in all cases of UPD in this study that were not complete isodisomy. b: SNP Microarray software showing copy neutral LCSH in case #29. The large green LCSH blocks are separated by a region of heterozygosity. The allelic segregation (top) shows absence of AB alleles on the center line. Small breaks in the LCSH blocks are caused by repeat rich regions with no available SNPs. The acrocentric chromosomes have no short arm SNPs so this represents a telomeric LCSH that increases UPD association. The locations of two microsatellite markers are shown in yellow.
other chromosomes and one control had one 13 Mb run and two runs of 9 Mb, suggesting underlying low grade IBD in each. The patients with qualifying LCSH that were not UPD, had a slightly higher overall second longest run average with 2/16 cases having multiple runs of 9 Mb. The latter indicates that low grade IBD has the potential to reach the qualifying length criteria at the interstitial threshold set for this study, generating false positives in at least a few cases.
DISCUSSION Control Selection The patient controls used to establish the normal curve may be slightly shifted toward longer LCSH than the normal population and are likely to also include more IBD, as these carry incumbent higher risk for recessive disorders. The control patients are also likely to have trisomy rescue that doesn’t result in UPD, but is
Whole 1 Whole 1
Whole 2 Whole 6
Case # CHR #
(a) IsoUPD 1 1 2 1
2 6
6
6
7
7 8 8 14 15
15 15
X X
3 4
5
6
7
8 9 10 11 12
13 14
15 16
(b) Hetero-isoUPD 17 6 13–139.4 18 6 20.3–134; 156.8–170.9 (qter)
Whole X Whole X
Whole 15 Whole 15
Whole 7 Whole 8 Whole 8 Whole 14 Whole 15
Whole 7
Whole 6
Whole 6
LCSH interval (start–end)
126 127.8
155 155
100 100
158 146 146 106 100
158
170
170
243 170
247 247
LCSH total (mb)
1 yo 7 yo 2 months 3.5 yo 4 mo 9 yo 9 yo
RSSa AR/T RISK AR/T RISK AR/T/IS ASa PWSa ASa
TNDM AR/T RISK
6 months 3.25 yo
5 yo 28 yo
2.4 yo
RSSa
FraX x2 FraX (premut)x2
NB
NB
16 months IUD 20 wks
7 yo 6 yo
Age
TNDM/TRISOMY 6
TNDM
AR/T RISK AR/T RISK þ IS
AR/T RISK AR/T RISK
Associated syndrome
pat-2 mat-2
mat pat
mat pat
mat pat unk mat pat
mat
pat
pat
pat unk
unk pat
Origin
PRO PRO
RET RET
PRO PRO
PRO PRO PRO PRO PRO
PRO
PRO
PRO
PRO PRO
PRO PRO
ASC
b
20 34
avg 28.7
31 22
34 39
29 33 40 31 19
29
32
30
25 21
16 NA
MAc at delivery
TABLE (Continued)
TNDM/severe IUGR Osteopenia, Hx placental insufficiency, recurrent, Infections, no dysmorphism
Mild ASD and hyperactive. Neonatal seizures, extreme hypotonia, profound MR, extreme cortical blindness. N/A IUGR, shortened long bones, left club foot, wide toe space left side, micrognathia, mild edema of the posterior skull and neck, small stomachpossible hypotelorism, lemon shaped head and achogenic area posterior to the heart. Oligohydraminos, hydrops fetalis with echocardiographic findings of the pulmonary stenosis and hypoplastic right heart. The newborn had whole body edema. Low grade mosaic trisomy 6 in amniocytes. Premature birth (30 wks), urethral stenosis, DD. N/A Aspergers, ADHD. Failure to thrive. Icthyosis, mild DD. Large tongue, increased ht/wt, creases behind ears, ears assymetric, nevus. MR, lack of coordination. Ataxia, ocular apraxia, periatrial white matter, seizures, CP. MR and Klinefelter syndrome. Clinically normal with recurrent miscarriages.
Clinical symptoms
TABLE I. SNP Microarray Detected UPD Cases (a–c). (a) Combined Retrospective (2) and Prospective (14) Cases of isoUPD, (b) Combined Retrospective (6) and Prospective (13) Cases of Hetero-isoUPD, (c) Two Segmental UPD Cases. A High Incidence of LCSH Telomeric Involvement (Red) can be Observed in Both Hetero/isoUPD and Segmental UPD
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15 15 15 15
16 18 21
29 30 31 32
33 34 35
(pter) 18.3–31.9; 60.4–89 52.6–67; 95–100.2 (qter) 38.2–68.2 (pter) 18.3–27, 92.8–100.4 (qter) 0 (pter)–14.4; 64.9–88.7 7–68.9 20.5–35.4
0 (pter)–4; 27.2–71 118.7–132.2 (qter) 40.2–76.1 34.9–46.1; 80.9–91.9 50.8–95.3
29.8
70.8 30.0
avg 46.3 SD 33.57
38.2 61.9 14.9
42.3 19.6 30.0 16.3
47.8 13.5 35.9 22.2 44.5
40.6 23.2 87.7
39.2 48.4
LCSH total
FraX x2
AR RISK BWS
NB
12 yo 11yo
AF 3 yo 5 yo
N/A 5 yo NB 17
PWSa PWSa PWSa PWSa AR/T RISK AR/T RISK AR/T RISK
AF AF 2 yo N/A 15 yo
5 months 1.5 yo 6 months
RSSa RSSa partial tri/ tetrasomy AR/T RISK AR/T RISK PWSa PWSa PWSa
AF 9 yo
Age
AR/T RISK RSSa
Associated syndrome
seg-mat
seg-mat seg-pat
mat-1 pat-2 mat-1
mat-2 mat-1 mat-1 mat-2
mat-2 mat-1 mat-1 mat-1 mat-1
mat-2 mat-1 mat-2
mat-1 mat-2
Origin
RET
PRO PRO
RET PRO PRO
RET PRO PRO PRO
RET RET PRO RET PRO
PRO PRO PRO
RET PRO
ASCb
Encephalopathy. Ompthalocele, hemihypertrophy, bilateral Wilm’s tumor. Fragile X full mutation, no paternal FMR1 allele.
Mosaic CVS, Delayed speech, overlapping 4th fingers. DD, congenital anomaly of nervous system.
Failure to thrive. Unknown metabolic disorder Prominent forehead, plagiocephaly, hypertelorism, low set ears, short toe, inside ear prominent, pneumothorax. Mosaic CVS Mosaic CVS N/A N/A Congenital anomalies of muscle tendon fascia. N/A N/A Hypotonia, poor feeding. N/A
N/A Short stature
Clinical symptoms
33
25 21
avg 35.6
39 38 38
43 NA NA 39
42 41 36 42 42
NA 25 21
40 19
MAc at delivery
pter, p terminus; qter, q terminus; PWS, Prader-Willi Syndome; AS, Angelman syndrome; AR/T, autosomal recessive/trisomy; IS, imprinting syndrome; NB, newborn; TNDM, Transient Neonatal Diabetes; FraX, Fragile X; RSS, Russell Silver Syndrome; unk, unknown; mat, maternal; pat, paternal; mat1, maternal meiosis I; mat-2, maternal meiosis II; pat-2, paternal meiosis II; seg-mat, segmental maternal UPD; pro, prospective; ret, retrospective; ASD, atrial septal defect; ADHD, attention deficit hyperactivity disorder; FTT, failure to thrive; MR, mental retardation; IUGR, interuterine growth retardation; SS, short stature; DD, developmental delay; CP, cleft palate; BWS, Beckwith-Weidemann Syndrome; yo, years old; mo, months old; wks, weeks. a Additional autosomal recessive and trisomy risk. b ASC: ascertainment. c MA: Maternal age.
X
125.1–154.9 (qter)
9 12 15 15 15
24 25 26 27 28
38
7 7 8
21 22 23
0 (pter)–9.3; 78.3–108.2 0 (pter)–7.8; 55.1–75.7; 138.8–158.8 (qter) 51.7–92.3 135.4–158.8 (qter) 0 (pter)–76.4; 134.8–146.1
63.5–134.3 (qter) 0.2 (pter)–30.2
7 7
19 20
LCSH interval (start–end)
(c) Segmental UPD 36 11 37 11
CHR #
Case #
TABLE I(Continued)
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FIG. 4. a: Case #2, example of a complete isoUPDpat1, (b) Case #22, hetero-isoUPD8mat associated with 1–2 ring markers of that chromosome, (c) Case #25, hetero-isoUPD 12mat the smallest UPD associated LCSH in the study(13.5 Mb) with UPD correlating telomeric location, (d) Case #19, hetero-isoUPD 7mat; 39.2 Mb LCSH total with telomere block, (e) Case #5, isoUPD 6pat in a patient with TNDM, (f) Case #18, hetero-isoUPD6mat, (g) Case #38, segmental UPD X in a neonate with two copies of the maternal full FMR1 mutation, (h) Two fraternal brothers with the common ‘‘false positive’’ LCSH region on chromosome 11; both inherited the same pair with recombination evident in the short arm by the novel HZ block.
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FIG. 5. a: Case #36: Segmental UPD 11qmat showing telomere association of LCSH. Microsatellite markers show isoUPD and biparental regions. b: Microsatellite evidence of segmental UPD is depicted by the paternal allele at about 10% the intensity of maternal allele with equal intensity of alleles in a heterozygous region marker. c: Case #37: Mosaic segmental UPD 11p that was associated with a classic Beckwith-Weidemann phenotype. The mix of AB (germline) alleles with converted AA or BB alleles at each SNP creates novel mosaic tracts that represent the relative percentage of segmental UPD. Note the copy neutral dosage. Reduced (50%) maternal methylation of LIT1 was found in subsequent molecular analysis. At a slightly higher percentage of UPD the LCSH block would appear, depending on the algorithm setting.
susceptible to the early trisomy developmental effects. However, this control group is needed, because it is from patient analysis that we are attempting to select candidates for UPD follow-up testing without running trios on all patients. A recent study [McMullan et al., 2009] documented runs of HZ in trios using SNP arrays, supplying a list of these regions. They found no difference in 120 patients with mental retardation and their parents as far as the incidence or length of homozygous regions, although a couple of cases of multiple chromosomal LCSH 10 Mb were noted and attributed to variable degrees of consanguinity. They found that the centromeric regions of chromosomes 11, 16, and 19 showed the most common single chromosomal LCSH 10 Mb, ranging between 10 and 19 Mb.
Retrospective/Prospective Studies The primary objective of these studies was to establish that UPD can be effectively identified using a SNP microarray study of a patient without requiring a parental trio analysis. A distinct pattern of HZ offering a UPD based ‘‘signature’’ was suggested by previous limited marker microsatellite studies of 36 cases of UPD which showed that most cases of UPD demonstrate at least partial isoUPD. The regional transitions from hetero to isoUPD correlate with meiotic recombination, which results in contiguous stretches of HZ that significantly exceed the largest single LSCH in the general patient population (average 3.64 Mb). In some cases, additional long runs
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TABLE II. Chromosome and Linear Positions of LCSH in 16 Positive Cases in which UPD was not confirmed. Note Absence of Telomeric Association and Frequent Centromeric Involvement (Red) Biparental LCSH positive cases CHR 2 2 3 3 3 4 5 6 7 7 8 11 11 15 16 18 Average
Start–stop 107.7–129 91.4–106 88–123 155.2–186.2 76.4–100 104.6–157 31.7–62.8 116.4–146 61.1–77.7 50.1–75.4 85.91–103.3 37.8–62 36.9–70.2 68.4–90.5 12.5–56.5 44.6–61.3
LCSH 20.8 14.7 35 30 23.6 52.8 31.1 29.5 16.6 25.3 17.4 24.2 33.3 22.1 44 16.7 26.183
Bands q12.3 ! q14.3 p11.1 ! q12.2 p11.2 ! q21.1 q25.2 ! q27.1 p12.3 ! q12.2 q24 ! q32.1 p13.3 ! q12.1 q22.1 ! q24.3 q11.1 ! q21.11 p12.2 ! q11.23 q21.2 ! q22.3 p12 ! q12.3 p12 ! q13.3 q23 ! q26.1 p13.3 ! q13 q21.1 ! q22.1
of HZ can be seen within the UPD chromosome consistent with additional meiotic recombination, preceding non-disjunction and post zygotic trisomy rescue. Short HZ blocks (1–3 Mb) may also be seen in heteroUPD regions that correlate with relative grandparental ‘‘background’’ HZ. Eight of the 38 patients with UPD in this report were studied on the SNP array following UPD confirmation as part of the original retrospective study. This cohort was important to: (1) determine how many were complete isochromosomes, (2) to establish the average size, range and pattern of HZ for prospective studies, and (3) to determine how many, if any, fail to exhibit a HZ stretch. All of the retrospective cases either demonstrated complete HZ (2/9) or HZ stretches of 13.5–42.2 Mb (7/9). Using the 13.5 Mb LCSH size as a prospective threshold for reporting possible UPD, 92 patients met the criteria. Of these, 46 were pursued for parental molecular confirmation (most involved chromosomes with imprinting) and 29 were confirmed as having whole or partial UPD.
LCSH Correlations Single chromosomal LCSH unrelated to whole chromosomal UPD may be due to a variety of reasons: genomic regions with low meiotic recombination, mitotic recombination (true segmental UPD), or low level autozygosity, all of which were observed in this study. It may be reasonable to anticipate that false negatives will be found with this signature, although none were found in the retrospective case studies, consistent with meiotic recombination occurring in most cases that are not complete isodisomy. In comparing the six retrospective and 13 prospectively identified cases of combined hetero/iso UPD with the 16 cases with qualifying LCSH that failed to
molecularly confirm, a strong correlation of telomeric involvement of LCSH with UPD can be seen in the positive group (13/19, including both ends in two cases) that was not present in the false positive cases. This is consistent with the most common maternal M1 non-disjunction and trisomy rescue model with a single crossover in one chromosome arm. A false positive LCSH, on the other hand, is less likely to occur at a high recombinational telomeric site, which may account for this difference. Comparing the two groups, the false positive cases had: a shorter average LCSH (26.18 Mb vs. 46.32 Mb), a greater frequency of centromeric involvement that might be expected from this region of reduced recombination, and no telomeric association. The power of telomeric LCSH UPD association was tested by one case late in this study that ‘‘qualified’’ only by total size of two small runs of HZ at each end of chromosome 15 (8.7 and 7.6 Mb) and was indeed clinically and methylation positive for PWS. This suggests that separate thresholds for interstitial and telomeric LCSH may be optimal for UPD association. The question of whether different qualifying thresholds should be applied based on the relevant chromosomal size is reasonable. Clearly the opportunity for a recombination above the threshold is reduced in the smaller chromosomes so some compensation for size is reasonable for optimal correlation. Because the smallest chromosomes are unlikely to recombine 2 (minimizing the qualifying opportunity on either end), reducing the threshold for smaller chromosomes proportionally even below the 10 Mb level may be justified. It was important for UPD correlation that the LCSH be on a solitary chromosome and not be accompanied by other runs of HZ on additional chromosomes that are characteristic of autozygosity. As both the number and length of LCSH increase with the degree of consanguinity, single runs of HZ due to distant common descent appear to be so reduced by meiotic recombination that the maximum LCSH length is usually well below the suggested threshold for UPD. Therefore, any LCSH over 10 Mb on a second chromosome was considered exclusionary for reporting possible UPD in this study. Higher degrees of IBD result in longer LCSH, but these appear on many chromosomes and can easily be excluded as UPD associated. It remains to be determined what the percentages of false positive and negative UPD signatures will be when enough cases are tested to establish the best correlating LCSH threshold. It is unlikely that all cases can be detected even when the optimal threshold is established, since some cases of UPD without homologous recombination may be expected, although absence of meiotic recombination may be rare [Bugge et al., 1998]. A reduction in the false positive HZ runs may depend on factoring in the common inherited runs of HZ that exists in most populations. A good example of this is the apparent low recombination region around 13q21 that resulted in a high frequency of extended HZ (seven cases) at that site in our studies. The centromeric regions of chromosome 3 and 11 also revealed multiple false positive LCSH in our studies. Extended regions of HZ in LD are present at numerous genomic sites [Curtis et al., 2008], which can then predispose to a false positive UPD signature. There is no difference in the appearance of the UPD positive centromeric LCSH associated with M2 non-disjunction and UPD negative large centromeric runs so differentiating is very important. The 3 and 11 centromeric regions in 6b are unique as
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FIG. 6. a: Eight representative cases of UPD associated LCSH showing relative position of HZ and heterozygosity. b: Most common regions of HZ blocks not associated with UPD. Fraternal brothers with matching centromeric LCSH were noted for both the centromeric region of 11 and the 13q21 region. These regions were the two most common false positive ‘‘hot spots’’ which reached >15 Mb in multiple cases apparently due to common populationbased haplotypes. HZ stretches across the 11 centromere are particularly common.
common LCSH associated UPD negative regions, apparently related to low recombination frequencies. Other centromeres rarely show any runs (e.g., chrs. 14 and 15). An overlapping recombination frequency map could become a reasonable tool to derive LDbased UPD probability by calculating the appropriate statistical significance of homozygotic runs. The study of Bruce offers support of our conclusions [Bruce et al., 2005]. They looked at six patients with UPD7 and identified regions of iso and heterodisomy in a retrospective SNP array trio study. Using their recombination sites, a LCSH association can be seen in all six cases (2-isoUPD, 3-hetero-isoUPD and one segUPD). The hetero-iso cases had a 47 Mb interstitial and a 19 Mb terminal LCSH in one case, a 25 Mb terminal and 41 Mb interstitial LCSH in another, and two interstitial LCSH (27 and 26 Mb) in the third. The segmental case had a 35 Mb terminal LCSH. Additional confirmation is available from Conlin et al. [2010] who focused on the mixed allele patterns of disomic and trisomic cell lines in mosaic trisomies by SNP array analysis and were able to identify the origin of the mosaicism. They identified five mosaic trisomies with meiotic origin; three of these were considered UPD in the disomic line by virtue of 1–3 crossover events with associated LCSH and trisomy. Four more cases with no evidence of trisomy and LCSH >20 Mb were also considered UPD due to the apparent meiotic recombination related LCSH origin and absence of IBD related LCSH on other chromosomes, although only two of the seven had parental confirmation. All but one of these cases displayed telomeric LCSH.
Segmental UPD Mitotic recombination leading to segmental UPD is, by definition, a copy number neutral post zygotic event which can occur at various time points in gestation, as reflected by the three cases described in this report. Cases 36 and 38 showed the UPD associated LCSH, consistent with mitotic recombination in early development. However, only the latter had complete parental exclusion in subsequent microsatellite analysis and, therefore, recombination must have occurred very early in embryogenesis. Conversely, the BWS patient (case 37) showed distinct copy neutral mosaic heterozygote allele patterns, which aligned midway between the homozygote (AA and BB) allele tracts and the usual heterozygote (AB) line (Fig. 5c). The position of these tracts, a mix of germ line AB SNPs and ‘‘converted’’ AA or BB SNPs, supplies a relative dosage of DNA with segmental UPD (about 50% in this patient). Note that it is the relative dosage of the A to B allele that is being depicted at each point (SNP) in the tract. The initiating mitotic recombination event is, therefore, likely to have taken place later in development in this patient compared with the other two patients. The presence of the residual germ line generated a unique mosaic heterozygote allele pattern in the software that is highly recognizable, although without an accompanying LCSH. All three of these cases demonstrated telomere association, which theoretically may be expected if mitotic recombination represents DNA repair occurring secondary to a double strand break, which is the generally favored mechanism. Previous studies confirm the
PAPENHAUSEN ET AL. strong correlation of segmental UPD with telomeric regions [Kotzot, 2008]. Molecular follow-up for UPD of cases with qualifying LCSH should include targeting of the HZ interval to rule out segmental UPD. When copy neutral mixed allele tracts are present, quantitative analysis is recommended.
Additional Risk Few of the patients with LCSH 13.5 Mb in non-imprinted chromosomes were followed-up for parental confirmation of UPD. It is likely that the regional HZ related recessive allele risk was thought to exist regardless of whether the etiology was through a UPD pathway. However, the common trisomy rescue UPD etiology does have an implicit risk of trisomy effects in early development. A good example of possible occult trisomy 18 effects is case 35 which had a phenotype with characteristic overlapping fingers. Additional evidence of trisomy rescue are found in three cases (23, 25, and 32) which showed placental trisomy and one case (6) amniotic fluid trisomy. Molecular confirmation of UPD was confirmed in all 13 patients pursued of the 16 cases with complete HZ. This level of LD makes UPD a virtual certainty; so confirmation is probably not necessary in practice, except to determine the parent of origin, particularly important for imprinted regions. The three cases that were not pursued for parental origin involved non-imprinted chromosomes.
Etiology The large excess of maternal origin (17 of 19) of mixed hetero/ isoUPD found in this study is consistent with previous studies [Kotzot, 2008] and the much higher maternal versus paternal meiotic non-disjunction rates. M1 non-disjunction with post zygotic trisomy rescue results in UPD with centromeric heterodisomy and distal isodisomy, the reverse of M2 derived cases. Complete isodisomy, on the other hand, must result from a post -zygotic (somatic) pathway of monosomy rescue or rescue from a post zygotic derived trisomy. It is of interest that most cases of transient neonatal diabetes mellitus (TNDM) appear to correlate with paternal isoUPD (two of three in this study) and not mixed hetero/isoUPD [Eggermann et al., 2001], supporting a separate pathway to paternal UPD reflective of the paucity of non-disjunction in spermatogenesis. There was an 8:5 paternal predominance in the thirteen cases of total isoUPD with origin established in this study. Isodisomy has a high correlation with paternal UPD [Zlotogora, 2004; Kotzot, 2008], which in many cases may be due to monosomy rescue. The rarity of evidence of mosaic placentas in isodisomy cases offers support for that hypothesis and against somatic generation followed by rescue from trisomy, although low level amniocyte trisomy was clear evidence for the latter in case 6. Trisomy rescue has been suggested to be more likely due to the expected longer survival of trisomic zygotes compared with monosomic counterparts. However, the high incidence of blastomere monosomy, secondary to maternal non-disjunction, offers ample opportunity for a single step generation of complete isodisomy as opposed to the two-step generation and rescue from trisomy. If monosomy rescue is then the primary pathway to paternal isoUPD, an increased maternal age for paternal cases relative to maternal
767 cases then may be expected. However, the average maternal age at birth of eight cases in this report with isoUPDpat were not significantly different from the five isoUPDmat cases with available maternal age, consistent with age independent post-zygotic trisomy generation and rescue. Clearly more cases are needed to offer insight into the primary mechanism for isoUPD. In regard to which chromosomes are more likely to be detected as complete isochromosomes, additional factors may contribute such as size (relating to recessive allele risk) and incidence of the chromosome in non-disjunction, as well as rescue selection pressure. Maternal UPD 7 has been thought to have an excess of isodisomy [Mergenthaler et al., 2000], but only two of six maternal UPD7 cases reported in this study showed isodisomy. The UPD mechanism for the two whole isodisomy fragile X cases appears distinctly different. The absence of X recombination in the XXY case is most consistent with a 46,XY zygote with somatic nondisjunction, yielding 47, XXY and the nonviable 45,Y (similar to the isoUPD6 derivation). The isoUPDpat etiology for the XX case may best fit combined paternal and maternal meiotic non-disjunction events (two X’s and no X, respectively), since it seems likely that there would be little developmental selective pressure for monosomy or trisomy rescue in an X or XXX fetus.
Summary A characteristic single chromosome-based LCSH >13.5 Mb was present in all 19 of our prospective and retrospective detected mixed hetero/isoUPD cases (one patient had a LCSH at each end in a chromosome 15 that added together equaled 16.3 Mb). Forty-two percent of the confirmed UPD cases showed complete isodisomy (16/38). Four cases were detected after trisomy was noted in CVS/ amniocyte testing and one case showed centromeric trisomy in conjunction with a supernumerary marker of that chromosome, both of which are recognized cytogenetic associations with UPD indicative of the ancillary trisomy risk. Very few cases with qualifying LCSH present in non-imprinted chromosomes were pursued in these studies. Although the risk of recessive disorders would exist for all genes in the HZ interval, regardless of the etiology, the risk of the early developmental effects of occult trisomy removed by negative selection accompanies the most common UPD etiology of trisomy rescue. Therefore, molecular testing for UPD should be considered when the associated phenotype is consistent with mosaic trisomy of the LCSH bearing chromosome. Whole isochromosome UPD cases arising from monosomy rescue would bear no occult trisomy risk, but cannot easily be differentiated from those cases originating from trisomy rescue. The prevalence of telomere LCSH association with both complete chromosomal and segmental UPD supports use of a correlating threshold of approximately 20 Mb interstitially and 10 Mb (or less) telomeric.
NOTES ADDED IN PROOF Since the submission of this manuscript, 28 additional cases of confirmed UPD have been detected by the array signature. The complete isoUPD total has increased to 30, including a second case with concommittant mosaic trisomy, consistent with a postzygotic trisomy origin and rescue. The hetero-isoUPD total has increased to
768 32 and one more patient with segmental UPD of 11p and BWS has been confirmed.
REFERENCES Brown WT, Houck GE Jr, Jeziorowska A, Levinson FN, Ding X, Dobkin C, Zhong N, Henderson J, Brooks SS, Jenkins EC. 1993. Rapid fragile X carrier screening and prenatal diagnosis using a nonradioactive PCR test. JAMA 270:1569–1575. Bruce S, Leinonen R, Lindgren CM, Kivinen K, Dahlman-Wright K, Lipsanen-Nyman M, Hannula-Jouppi K, Kere J. 2005. Global analysis of uniparental disomy using high density genotyping arrays. J Med Genet 42:847–851. Bugge M, Collins A, Petersen MB, Fisher J, Brandt C, Hertz JM, Tranebjaerg L, de Lozier-Blanchet C, Nicolaides P, Brondum-Nielsen K, Morton N, Mikkelsen M. 1998. Non-disjunction of chromosome 18. Hum Mol Genet 7:661–669. Conlin LK, Thiel BD, Bonnemann CG, Medne L, Ernst LM, Zackai EH, Deardorff MA, Krantz ID, Hakonarson H, Spinner NB. 2010. Mechanisms of mosaicism, chimerism and uniparental disomy identified by single nucleotide polymorphism array analysis. Hum Mol Genet 19: 1263–1275. Creau-Goldberg N, Gegonne A, Delabar J, Cochet C, Cabanis MO, Stehelin D, Turleau C, de Grouchy J. 1987. Maternal origin of a de novo balanced t(21q21q) identified by ets-2 polymorphism. Hum Genet 76:396–398. Curtis D, Vine AE, Knight J. 2008. Study of regions of extended homozygosity provides a powerful method to explore haplotype structure of human populations. Ann Hum Genet 72:261–278. Eggermann T, Marg W, Mergenthaler S, Eggermann K, Schemmel V, Stoffers U, Zerresa K, Spranger S. 2001. Origin of uniparental disomy 6: presentation of a new case and review on the literature. Ann Genet 44:41–45. Engel E. 1980. A new genetic concept: Uniparental disomy and its potential effect, isodisomy. Am J Med Genet 6:137–143. Gibson J, Morton NE, Collins A. 2006. Extended tracts of homozygosity in outbred human populations. Hum Mol Genet 15:789–795. Kosaki K, McGinniss MJ, Veraksa AN, McGinnis WJ, Jones KL. 1997. Prader-Willi and Angelman syndromes: Diagnosis with a bisulfitetreated methylation-specific PCR method. Am J Med Genet 73:308–313. Kotzot D. 2008. Complex and segmental uniparental disomy updated. J Med Genet 45:545–556.
AMERICAN JOURNAL OF MEDICAL GENETICS PART A Li LH, Ho SF, Chen CH, Wei CY, Wong WC, Li LY, Hung SI, Chung WH, Pan WH, Lee MT, Tsai FJ, Chang CF, Wu JY, Chen YT. 2006. Long contiguous stretches of homozygosity in the human genome. Hum Mutat 27:1115–1121. McMullan DJ, Bonin M, Hehir-Kwa JY, de Vries BB, Dufke A, Rattenberry E, Steehouwer M, Moruz L, Pfundt R, de Leeuw N, Riess A, Altug-Teber O, Enders H, Singer S, Grasshoff U, Walter M, Walker JM, Lamb CV, Davison EV, Brueton L, Riess O, Veltman JA. 2009. Molecular karyotyping of patients with unexplained mental retardation by SNP arrays: A multicenter study. Hum Mutat 30:1082–1092. McQuillan R, Leutenegger AL, Abdel-Rahman R, Franklin CS, Pericic M, Barac-Lauc L, Smolej-Narancic N, Janicijevic B, Polasek O, Tenesa A, Macleod AK, Farrington SM, Rudan P, Hayward C, Vitart V, Rudan I, Wild SH, Dunlop MG, Wright AF, Campbell H, Wilson JF. 2008. Runs of homozygosity in European populations. Am J Hum Genet 83:359–372. Mergenthaler S, Wollmann HA, Burger B, Eggermann K, Kaiser P, Ranke MB, Schwanitz G, Eggermann T. 2000. Formation of uniparental disomy 7 delineated from new cases and a UPD7 case after trisomy 7 rescue. Presentation of own results and review of the literature. Ann Genet 43:15–21. Papenhausen PR, Tepperberg JH, Mowrey PN, Gadi IK, Shah HO, Sherman J, Pulijaal V, Nitowsky H, Sachs G, Lin JH. 1999. UPD risk assessment: three cytogenetic subgroups. Am J Hum Genet 65. Reboul MP, Tandonnet O, Biteau N, Belet-de Putter C, Rebouissoux L, Moradkhani K, Vu PY, Saura R, Arveiler B, Lacombe D, Taine L, Iron A. 2006. Mosaic maternal uniparental isodisomy for chromosome 7q21qter. Clin Genet 70:207–213. Robinson WP, Wagstaff J, Bernasconi F, Baccichetti C, Artifoni L, Franzoni E, Suslak L, Shih LY, Aviv H, Schinzel AA. 1993. Uniparental disomy explains the occurrence of the Angelman or Prader-Willi syndrome in patients with an additional small inv dup(15) chromosome. J Med Genet 30:756–760. Robinson WP, Bernasconi F, Basaran S, Yuksel-Apak M, Neri G, Serville F, Balicek P, Haluza R, Farah LM, Luleci G., et al. 1994. A somatic origin of homologous Robertsonian translocations and isochromosomes. Am J Hum Genet 54:290–302. Schollen E, Grunewald S, Keldermans L, Albrecht B, Korner C, Matthijs G. 2005. CDG-Id caused by homozygosity for an ALG3 mutation due to segmental maternal isodisomy UPD3(q21.3-qter). Eur J Med Genet 48:153–158. Zlotogora J. 2004. Parents of children with autosomal recessive diseases are not always carriers of the respective mutant alleles. Hum Genet 114: 521–526.
