UPD AND TFM AFTER DETECTION OF MOSAICS IN CVS: CHROMOSOME SPECIFIC RISKS FROM 52.673 DIAGNOSES

INTRODUCTION

AIM OF THE STUDY

Chromosome mosaicism is the bane of cytogenetics prenatal diagnosis (PD). It’s detected in ~1–2% of chorionic villi samples (CVS) and can involve different numerical and structural chromosome abnormalities and feto-placental cell lineages1-5. Most time it turns out to have been a false alarm such as in the case of confined placental mosaicism (CPM) 6. It can be caused by a mitotic error in a normal conceptus or by a meiotic error with a subsequent mitotic event generating a normal cell line. Trisomy rescue of an abnormal conceptus can also bear to uniparental disomy (UPD) in the fetus7,8. The distribution of the normal and abnormal cell lines in fetus and in placenta depends on the time and place of the mitotic event. To define if mosaicism are CPM or generalized to fetus (TFM) a confirmatory amniocentesis is advisable, although Daniel et al.9 have reported that 10% of CVS interpreted as CPM may reflect a cryptic fetal mosaicism which might or might not have phenotypic consequence.

We report a retrospective survey of 52.673 consecutive prenatal diagnoses on CVS performed in a single center (TOMA laboratory) from 2000 to 2012. In this study we included 1136 mosaics on CVS, of these 886 were followed by amniocentesis with the aim to explore the risk of TFM and UPD in relation to: i) distribution of the abnormal cell line in placental tissues ii) chromosome abnormality iii) chromosome involved.

All cases of mosaicim on CVS, the number of cases followed by AF and all TFM cases detected are reported in Table 1. The 886 cases followed by AF were classified into six classes of mosaicism on the basis of tissue involved (Figure 1) and the relative frequencies are reported in Table 2. The risk of fetal involvement also considering mosaic (MA) and non mosaic abonormalities (NMA) are reported in Table 3. Tables 4, 5, 6 and 7 report all 886 cases of CPM and TFM divided on the basis of

chromosome abnormalities. UPD was detected in 6 cases involving a proposed or documented imprinted chromosome, see Table 8.

General Frequenciesl The frequency of chromosome mosaicism on 52673 CVS combining STC and LTC was 2.16%, similar to the frequencies reported by other authors12. Nowadays not combining the two methods this percentage was redefined as 1%. l The general risk of fetal involvement (TFM) was 12.7%, comparable with EUCROMIC one (10.4%). This value can decrease or increase on the basis of the distribution of the abnormal cell line in placental tissues (Table 3).l The difference between TOMA and EUCROMIC studies, as shown in table 3, are the risk of TFMIV (4.35% vs. 0%) and percentage of TFMV in non mosaic state (30.5% vs. 83.3%). These differences could be caused by the different sample size (322 vs. 93 cases for type I CVS mosaicism and 426 vs. 55 cases for type II) and by the fact that our was a mono-centric survey based on homogenous criteria.

Autosomal trisomiesl The most common autosomal trisomies confirmed at amniocentesis were those of chromosome 21 (32%, 16/49 cases) and 18 (17%, 8/46 cases).l Trisomies 2, 3 and 7 were the most common autosomal trisomies detected on CVS but they were never confirmed at amniocentesis. In addition we never observed upd7, one cases of upd7 was reported in the literature by Kalousek et al. (1996)13. UPD investigation and detailed US examination are suggested in trisomy 7 CVS mosaic cases even if the risk for UPD is very low.l Trisomy 13 was frequently observed in CVS but rarely confirmed in AF (2.6%, 1/39 cases) as reported by other authors.l We observed a TFM case with trisomy 4. To our knowledge only other four cases are reported.l Combining TOMA and EUCROMIC data trisomy 21 mosaicism on CVS is the most likely to represent a TFM ; a risk applies also with trisomies 4, 8, 9, 12, 13, 16, 18, 20.

Sex Chromosomes aneuploidiesl Mosaic monosomy X is the most frequent abnormality

detected on CVS with a risk of confirmation of 28% (28/101 cases).l X chromosome in mosaic condition was always confirmed as TFM (6 cases).l After detecting a mosaic 47,XXY karyotype on CVS, the risk of TFM was 35% (6/11 cases).

Structural Chromosome abnormalities and markersl The highest frequency of TFM was detected in the karyotype with mosaic supernumerary marker chromsome: 35% (19/54) with similar frequency of sat and no sat confirmed at amniocentesis. l The risk of TFM in the presence of 46,unbalanced rearrangement is low 5%, while among TFM cases with a 46, balanced rearr karyotype two involved a robetsonian translocation and two a reciprocal translocation.

Polyploidies and multiple trisomiesl Only triploidies were confirmed on AF, while tetraploidies were always CPM. There are rare clinical reports of fetuses/infants with mosaic tetraploidy, consequently we suggest to consider this finding only in the presence of ultrasound abnormalities.l In the presence of multiple autosomal trisomies with normal cell line as reported by Pertile (personal communication, 2002) a fetal involvement is never seen.

UPD riskl UPD was detected in 6 cases (2.35%) involving chromosomes 14, 15 and 16. l 5/6 UPD cases were CPM.l The two cases of UPD14 were both CPMI with a low level of trisomy 14 in the cytotrophoblat. Consequently this cases were be undetected if STC were not harvested or if analysis was performed combining LTC and QF-PCR for common aneuploidies, as most laboratories now offer. The indication for prenatal diagnosis was advanced maternal age.

Chromosome analyses were performed in agreement with the Italian Guidelines which are consistent with the European ones. Standard protocols were used to set up the cultures and chromosome preparations, the applied banding techniques were QFQ; karyotype was formulated following the ISCN10 indications progressively updated during the survey period. CVS analysis was performed combing short-term culture (STC) and long-term culture (LTC), while for amniotic fluid (AF) samples in situ cultures were harvested. A condition of mosaicism was defined as the presence of at least two cells with the same numerical or structural chromosome alteration. TFM was defined as the presence of at least one colony showing the same abnormality previously observed in CVS. UPD was explored in all cases involving a proposed or documented imprinted chromosome (chromosomes 2, 6, 7, 11, 14, 15, 16 and 20) by microsatellite segregation analysis from parents to fetus. The present survey includes also all cases previously reported by Grati et al.11 and the data are presented in the same format of EUCROMIC study12 to facilitate the comparison.

RESULTS

CONCLUSION

DISCUSSION

Despite of the majority of mosaicism identified at CVS does not presage an abnormal baby, we have presented the risk of TFM related to the type of chromosome abnormality detected and the distribution of the abnormal cell line in placental tissues. We think that our experience could be used as a reference during genetic counseling to the couple after detecting a mosaicism at CVS.

1 Vejerslev LO, Mikkelsen M. Prenat Diagn 1989; 9: 575– 588.2 Lancet 1991; 337: 1491– 1499.3 Ledbetter DH, Zachary JM, Simpson JL et al. Prenat Diagn 1992; 12:317– 345.4 Prenatal Diagn 1994; 14: 363– 379.5 Wolstenholme J, Rooney DE, Davison EV. Prenat Diagn 1994; 14:345– 361.6 Simoni G, Sirchia SM. Prenat Diagn 1994; 14: 1185–1189.7 Robinson WP, Barrett IJ, Bernard L et al. Am J Hum Genet 1997; 60: 917– 927.

8 Engel E, DeLozier-Blanchet CD. Am J Med Genet 1991; 40: 432– 439.9 Daniel A et al. Prenat Diagn 2004; 24: 524–536.10 Shaffer LG et al. ISCN Karger, 2013. 11 Grati FR et al. Eur J Hum Genet 2003;14:282-288.12 Hahnemann JM, Vejerslev LO. Am J Med Genet 1997; 70: 179– 187.13 Kalousek et al. Am J Med Genet 1996;65:348-52.

Figure 1 (From Kalousek et al., 1993)

Three types of mosaicism on CVS which can give rise to CPM or TFM.

In all three CPM types the mosaicism is confined to the placenta while in TFM the mosaicism is generalized to fetus.

I) CPMI and TFMIV, abnormal cytotrophoblast and normal chorionic stroma

II) CPMII and TFMV, normal cytotrophoblast and abnormal chorionic stroma;

III) CPMIII and TFMVI, abnormal cytotrophoblast and chorionic stroma.

REFERENCES

F. MALVESTITI1, B. GRIMI1, C. AGRATI1, S. MILANI1, A. DI MECO1, G. FRASCOLI1, A. TROTTA1, R. LIUTI1, A. RUGGERI1, F. DULCETTI1, F. MAGGI1, G. SIMONI1, F.R. GRATI1 1RESEARCH AND DEVELOPMENT, CYTOGENETICS AND MOLECULAR BIOLOGY, TOMA ADVANCED BIOMEDICAL ASSAYS, BUSTO ARSIZIO (VA)

MATERIALS AND METHODS

Via F. Ferrer 25/27 • 21052 Busto Arsizio (VA) Italy • Tel +39 0331 652911 • Fax +39 0331 652919 • www.tomalab.com • C.F. e P.IVA 00772010120 • CCIAA Varese Rea 155894 • Capitale Sociale i.v. € 250.000 • ISO 9001:2008 • ISO 15189: 2007 • SIGUCERT 2009

TOMA LAB (present study)

EUCROMIC(Hahnemann and Vejerslev, 1997)

Total CVS karyotyped 52673 92246Mosaicism on CVS 1136 (2,16%) 650 (1,5%)

Follow up at amniocentesis 886 192TFM 113(12,75%) 20 (10,4%)

Table 1: All cases of mosaicism on CVS detected by TOMA Lab and reported by EUCROMIC study

Table 2: Incidences of the different types of mosai-cisms (CPM and TFM) found after chrionic villous and amniocytes karyotyping from TOMA and EU-CROMIC studies

TYPE NATURE TROPHOBLAST (STC) MESENCHYME (LTC)

AMNIOCYTES RELATIVE FREQUENCIES (TOMA Lab)

RELATIVE FREQUENCIES (EUCROMIC)

I CPM Abnormal Normal Normal 34,76% (308/886) 43,2% (83/192)II CPM Normal Abnormal Normal 42,32% (375/886) 25% (48/192)III CPM Abnormal Abnormal Normal 10,16% (90/886) 16,1% (31/192)IV TFM Abnormal Normal Abnormal 1,58% (14/886) 0%V TFM Normal Abnormal Abnormal 5,76% (51/886) 3,6% (7/192)VI TFM Abnormal Abnormal Abnormal 5,42% (48/886) 6,8% (13/192)

Table 3: Probabilities of confirmation on am-niocytes of Mosaic or Non Mosaic abnormal cell line considering the different combinations of the affected placental tissues

TROPHOBLAST MESENCHYME CONFIRMATION (direct) (culture) TOMA EUCROMIC

A N 4,35% (14/322) 0% (0/93)MA N 2,90% 0NMA N 8,64% 0N A 11,97% (51/426) 12,7% (7/55)N MA 8,99% 4,10%N NMA 30,51% 83,30%A A 34,78% (48/138) 33% (11/33)MA MA 30,13% 23,50%NMA MA 26,83% 18,20%MA NMA 76,92% 100%NMA NMA 45,45%

A=Abnormal; N=Normal; MA=Mosaic Abnormality; NMA=Non Mosaic Abnormality

Autosome trisomy

CPMI CPMII CPMIII TFMIV TFMV TFMVI Total TFM freq

+ 1 1 1 + 2 5 57 2 64 + 3 24 2 26 + 4 2 1 1 4 1/4 + 5 2 2 + 6 1 1 2 + 7 36 25 4 65 + 8 10 15 1 2 28 2/28 + 9 3 9 3 15 + 10 4 7 11 + 11 3 1 4 + 12 1 9 1 11 1/12 + 13 21 10 7 1 39 1/39 (2%) + 14 4 3 3 10 + 15 14 7 3 24 + 16 3 8 5 2 18 2/18 + 17 2 2 + 18 10 25 3 6 2 46 8/46 (17%) + 19 1 1 + 20 10 8 5 2 25 2/25 + 21 8 22 3 1 8 7 49 16/49 (32%) + 22 4 2 2 8TOTAL 479 33/479 (7%)

Sex Chromosome aneuploidies

CPMI CPMII CPMIII TFMIV TFMV TFMVI Total TFM freq

47,XYY 1 1 2 1/245,X 37 25 11 7 11 10 101 28/101 (28%)

47,XXY 6 4 1 3 3 17 6/17 (35%)47,XXX 5 1 1 2 9 3/9

mos X/XX/XXX 6 6 6/6TOTAL 135 44/135 (32%)

Other abnorma-lities

CPMI CPMII CPMIII TFMIV TFMV TFMVI Total TFM freq

47,+mar 10 21 4 4 7 8 54 19/54 (35%)47,+der 2 2 1 5

46, rear or 45, Rob

47 74 2 5 4 132 9/132 (7%)

47,+i(13q) 3 2 1 6 1/647 +i(18q) 1 2 347,+i(7p) 3 3

Complex rearr 1 2 1 4 1/4- 22 1 1 2Other 1 3 1 5 4/5

Poliploidies CPMI CPMII CPMIII TFMIV TFMV TFMVI Total TFM freq

Triploidy 1 1 2 2/2Tetraploidy 25 11 20 56

TOTAL 58 2/58

Chromosome Ab-normalities

UPD No UPD/No of cases

Type of CPM or TFM

trisomy 2 2 0/62 -

trisomy 7 7 0/69 -

trisomy 6 6 0/2 -

trisomy 11 11 0/6 -

trisomy 14 14 2/10 2 (CPM I)

trisomy 15 15 1/28 1 CMPII

trisomy 16 16 3/17 2 (CPM III)+1(TFM VI)

trisomy 20 20 0/25 -

sSMC, others - 0/40 -

Total 259 6

Table 8

Table 7

Table 4

Table 5

Table 6

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