Journal of Obstetrics and Gynaecology

ISSN: 0144-3615 (Print) 1364-6893 (Online) Journal homepage: http://www.tandfonline.com/loi/ijog20

Prenatal diagnosis and molecular cytogenetic characterisation of a de novo 18p deletion G. Fogu, G. Capobianco, F. Cambosu, P. Bandiera, A. Pirino, M. A. Moro, P. M. Campus, G. Soro, M. Dessole & A. Montella To cite this article: G. Fogu, G. Capobianco, F. Cambosu, P. Bandiera, A. Pirino, M. A. Moro, P. M. Campus, G. Soro, M. Dessole & A. Montella (2014) Prenatal diagnosis and molecular cytogenetic characterisation of a de novo 18p deletion, Journal of Obstetrics and Gynaecology, 34:2, 192-193 To link to this article: http://dx.doi.org/10.3109/01443615.2013.834300

Published online: 23 Jan 2014.

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Acknowledgements We thank Christine O’Hara for her help with the English version of the manuscript. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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References Bédard E, Dimopoulos K, Gatzoulis MA. 2009. Has there been any progress made on pregnancy outcomes among women with pulmonary arterial hypertension? European Heart Journal 30:256–265. Bildirici I, Sumway JB. 2004. Intravenous and inhaled epoprostenol for primary pulmonary hypertension during pregnancy and delivery. Obstetrics and Gynecology 103:1102–1105. Bonnin M, Mercier FJ, Sitbon O et al. 2005. Severe pulmonary hypertension during pregnancy: mode of deliveryw and anesthetic management of 15 consecutive cases. Anesthesiology 102:1133–1137. Galiè N, Hoeper MM, Humbert M et al. 2009. Guidelines for the diagnosis and treatment of pulmonary hypertension. Task Force for Diagnosis and Treatment of Pulmonary Hypertension of European Society of Cardiology (ESC); European Respiratory Society (ERS); International Society of Heart and Lung Transplantation (ISHLT). European Respiratory Journal 34:1219–1263. Hill LL, De Wet CJ, Jacobsohn E et al. 2004. Peripartum substitution of inhaled epoprostenol for intravenous prostacyclin in a patient with primary pulmonary hypertension. Anaesthesiology 100:1603–1605. Humbert M, Morrell NW, Archer SL et al. 2004. Cellular and molecular pathobiology of pulmonary arterial hypertension. Journal of the American College of Cardiology 43:13S–24S. Kiely DG, Condliffe R, Webster V et al. 2010. Improved survival in pregnancy and pulmonary hypertension using a multiprofessional approach. British Journal of Obstetrics and Gynaecology 117:565–574. Monnery L, Nanson J, Charlton G. 2001. Primary pulmonary hypertension in pregnancy; a role for novel vasodilators. British Journal of Anaesthesia 87:295–298. Stewart R, Tuazon D, Olson G et al. 2001. Pregnancy and primary pulmonary hypertension. Successful outcome with epoprostenol therapy. Chest 119:973–975.

Prenatal diagnosis and molecular cytogenetic characterisation of a de novo 18p deletion G. Fogu1, G. Capobianco2, F. Cambosu1, P. Bandiera3, A. Pirino3, M. A. Moro1, P. M. Campus1, G. Soro3, M. Dessole2 & A. Montella3 1Clinical Genetics, Department of Biomedical Sciences,

is greatly variable and it may be not evident at birth, or during the pregnancy, even though some echographic signs, such as increased nuchal translucency (NT), may seem to be suggestive (Kim et al. 2004). Here, we describe a de novo case of 18p deletion prenatally ascertained. We report the findings of the autoptic examination, as well as of cytogenetic studies.

Case report Amniocentesis was performed at 17 weeks’ gestation on a 39-yearold gravida referred for prenatal diagnosis because of her maternal age. The woman had refused to undergo 1st trimester screening by combined test (nuchal translucency, PAPP-A and free-βhCG) and opted to go directly to amniocentesis. The woman’s husband was 42 years old. Both parents were healthy and not consanguineous. The family history was negative for congenital malformations and mental retardation. The couple had an 18-year-old daughter, in good health. The pregnancy was uncomplicated and all the laboratory tests were normal. At the time of the amniocentesis, the amniotic fluid alpha-fetoprotein level was lower than normal (8,666 ng/ml vs 9,500 ng/ml). Ultrasonographic examination at 18 weeks ⫹ 5, before the therapeutic termination of pregnancy showed: biparietal diameter and head circumference slightly reduced, normal limbs, normal thoracic, abdominal and pelvic organs. Our Ethics Committee approved this research.

Post-mortem examination The male fetus weighed 380 g. The external examination showed ipsicephaly, nuchal oedema, short neck and facial dysmorphic signs, including thick eyebrows with synophrys, broad nasal bridge, anteverted nares, little mouth with thin lips and micrognatia. The limbs were of normal length, with overlapping toes (the second toe over the third one and the fourth over the fifth) in the right foot (Figure 1). External genitalia were normal. At the autoptic examination, the right lung showed four lobes; all the other organs of thorax, abdomen and pelvis were normal. Cytogenetics and fluorescence in situ hybridisation Metaphase chromosome analysis was performed on cultured amniocytes. To this purpose, amniotic fluid, collected by amniocentesis, was centrifuged and a cell pellet was suspended in 12,5 ml of complete medium (Chang medium supplemented with glutamine, penicillin and streptomycin). Aliquots of 2.5–3 ml of cell suspension were inoculated into 30 mm Petri dishes with a coverslip at the bottom, then incubated at 37°C in 5% CO2 atmosphere. After 7–8 days, the cell growth was daily evaluated by inverted microscope, then at 8–10 days of culture, when the growth was optimal for number of clones and mitotic index, metaphases were blocked by colcemid treatment for 2 h and harvested according to the in situ

2Gynaecologic and Obstetric Clinic, Department of Surgical, Microsurgical and Medical Sciences and 3Department of Biomedical Sciences, Division of Human Anatomy, University of Sassari, Italy

DOI: 10.3109/01443615.2013.834300 Correspondence: G. Capobianco, Gynecologic and Obstetric Clinic, Sassari University, Viale San Pietro 12, 07100 Sassari, Italy. E-mail: [email protected]

Introduction Monosomy for the short arm of chromosome 18 is one of the most common autosomal deletion syndromes. Since the first report (Thieffry et al. 1963) of del(18p) in 1963, more than 150 cases have been reported (Jones 2006). The estimated incidence of this disorder is about 1:50,000 live-born infants, with a female to male ratio of 3:2 (Turleau 2008). Most cases are the result of de novo terminal deletions or, less frequently, of segregation of a balanced translocation in one of the parents. Approximately one in six of the reported patients have an unbalanced whole-arm translocation between one chromosome 18 and one of the acrocentric chromosomes, predominantly chromosome 21 or 22. The phenotype of this chromosome imbalance

Figure 1. Lateral view of the fetus. Inset: right foot with overlapping toes.

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Figure 2. FISH image of metaphase spread of the propositus and cut-out of maternal and paternal chromosomes 18. The arrow indicates the reduced centromeric signal on the deleted chromosome 18.

method. The medium was removed and replaced by hypotonic solution (KCl 0.075M/NaCitrate 0.5%) for 10 min at room temperature, followed by two passages in fixative methanol:acetic acid 3:1. The coverslips were then removed from the Petri dishes and allowed to dry at room temperature. The standard cytogenetic analysis was performed in QFQ banding on 10–12 clones from two or more independent cultures. Chromosome analyses of both parents were performed on blood lymphocytes, using standard methodologies. Fluorescence in situ hybridisation (FISH) experiments were done on amniocytes and on peripheral lymphocytes of both parents using a mix of subtelomeric18p probe (tel18p, red/green labelled), and a specific centromeric alphoid probe (CEP18, Aqua) (Totelvision Vysis, mix No. 11). The slides used for FISH, 1-day-old, were pre-treated with 2XSSC for 15 min at room temperature, dehydrated through a decreasing alcohol sequence and air dried. Probe mix and slides were co-denatured at 72°C for 3 min and then incubated overnight at 37°C using the Vysis Hybrite hybridisation system. Post-hybridisation washes, according to manufacturer’s protocol, were done with 0.4X SSC, 0.3% Nonidet at 72°C for 2 min, followed by a rapid wash in 2X SSC, 0,1% Nonidet at room temperature for 30 s. The slides were then drained, stained with DAPI/antifade and observed with fluorescence microscope Olympus BX-61, using suitable filters. Cytogenetic analysis of cultured amniocytes showed an abnormal male karyotype, with a chromosome 18 deleted of its apparently entire short arm. According to QFQ banding, the fetal karyotype could be indicated as 46,XY,18p-. The parents had normal chromosomes, indicating a de novo origin of del(18p). FISH analysis showed the absence of 18p telomeric signal on the deleted chromosome, indicative of a terminal deletion, and a centromeric spot of greatly reduced intensity and size in the same chromosome (Figure 2). According to FISH results, the fetal karyotype could be indicated as follows: 46,XY.ish del(18)(p10pter)(tel18p-, dim D18Z1). FISH experiments on parental chromosomes showed, in both parents, two normal chromosomes 18, with centromeric blocks of normal size (Figure 2). The phenotype of 18p deletion is greatly variable and not evident at birth. It can become recognisable at a later age, but it is not as characteristic as in other chromosomal syndromes. The phe-

notypic spectrum may include: round face; ptosis; flat and broad nasal bridge; wide mouth with short upper lip; small mandibles; irregular teeth and dental caries; large, protruding ears, often low and posteriorly rotated; short neck; pectus excavatum; kyphoscoliosis. Anomalies of the hands and of the feet may be present (Turleau 2008). The main malformations are holoprosencephaly (HPE), present in 10–15% of cases, which involves the abnormal development of the forebrain and the midface, cardiac defects and genital anomalies (Cohen 2006). Growth retardation, psychomotor delay and mental insufficiency, often mild, are constant symptoms of the syndrome. The fetal phenotype of 18p- is still more inconsistent, so the prenatal diagnosis of this chromosome imbalance usually represents an unexpected finding at villocentesis or amniocentesis. A report indicates the increased nuchal translucency (NT) as the only echographic finding at the 1st trimester. The association between fetal chromosomal abnormalities and increased NT is well established, mainly for trisomy 21 (Nicolaides et al. 1994), but there are limited experiences of other chromosomal anomalies. As for 18p monosomy, further reports would help to ascertain whether increased NT at the 1st trimester could be regarded as a constant finding in this chromosomal imbalance. In the present case, increased NT was not pointed out, even though the nuchal oedema seen at post-mortem examination was most likely connected to a misdiagnosed increased nuchal translucency. In the absence of echographic indications, and in mothers not at risk due to age, the pregnancy may reach term, with the birth of a baby who may be apparently normal or with minimal dysmorphic signs. As to cytogenetics, the case reported here shows an interesting aspect: a very faint FISH signal of the specific alphoid centromeric probe on the deleted chromosome 18. Subsequent FISH analysis with the same probe performed on both parents showed the presence, in each one of them, of two normal chromosomes 18, with a centromeric region equal in size in both homologues. These results allow us to conclude that the 18p deletion in the fetus included the entire short arm, with a deletion breakpoint lying inside the centromeric region. This may be consistent with a possible whole arm translocation between one chromosome 18 and another chromosome, likely occurring in maternal meiosis. These whole arm

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translocations usually involve one chromosome 18 and an acrocentric chromosome, frequently 21 or 22. The predominance of (18; acrocentric) translocations is likely due to some homologies between subsets of alpha satellite sequences at centromeric regions of chromosome 18 and of the acrocentric chromosome (Tharapel et al. 1991; Wang et al. 1997). In the present case, hypothetically, whole-arm translocation could have originated from both the chromosome 18 deleted of its short arm found in our patient, and a dicentric chromosome, which is the reciprocal translocation product, segregated in another gamete. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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References Cohen MM Jr. 2006. Holoprosencephaly: clinical, anatomic, and molecular dimensions. Birth Defects Research. Part A, Clinical and Molecular Teratology 76:658–673. Jones KL. 2006. Deletion 18p syndrome. In Smith’s recognizable pattern of human malformation. 6th ed. Philadelphia: Elsevier Saunders. p. 60–61. Kim YM, Cho EH, Kim JM et al. 2004. Del(18p) syndrome with increased nuchal translucency in prenatal diagnosis. Prenatal Diagnosis 24:161–164. Nicolaides KH, Brizot ML, Snijders RJ. 1994. Fetal nuchal translucency: ultrasound screening for fetal trisomies in the first trimester of pregnancy. British Journal of Obstetrics and Gynaecology 101:782–786. Tharapel AT, Qumsiyeh MB, Martens PR et al. 1991. Identification of the origin of centromeres in whole arm translocations using fluorescent in situ hybridization with alpha-satellite DNA probes. American Journal of Medical Genetics 40:117–120. Thieffry S, Arthuis M, de Grouchy J et al. 1963. Deletion of the short arms of chromosome 17–18: complex deformities with oligophrenia. Archives Francaises de Pediatrie 20:740–745. Turleau C. 2008. Monosomy 18p. Orphanet Journal of Rare Diseases 3:4–5. Wang JC, Nemana L, Kou S et al. 1997. Molecular cytogenetic characterization of 18;21 whole arm translocation associated with monosomy 18p. American Journal of Medical Genetics 71:463–466.

Conservative management of a spontaneous retroperitoneal haematoma following caesarean section secondary to an arteriovenous malformation: A rare case C. H. M. Bisseling, R. Hassan & H. C. Francis Department of Obstetrics and Gynaecology, University Hospital of Wales, Cardiff DOI: 10.3109/01443615.2013.835304 Correspondence: C. H. M. Bisseling, Department of Obstetrics and Gynaecology, University Hospital of Wales, Heath Park, Cardiff CF14 4XW, UK. E-mail: [email protected]

Introduction Retroperitoneal haemorrhage is a rare complication in pregnancy and there have been few cases reported in the literature. Often patients present with maternal collapse and the causes are variable (Rafi and Muppala 2010). Management of such cases can be very challenging. We report a case of a large retroperitoneal haematoma after caesarean section arising from an arteriovenous malformation (AVM).

Case report A 33-year-old multigravida underwent an elective caesarean and tubal ligation at 38 weeks’ gestation for four previous caesarean sections. She had been investigated for a connective tissue disorder because of a strong family history of vascular rupture. Ehlers–Danlos syndrome was considered but her collagen was normal. Although

some of the features fitted with Loeys–Dietz syndrome, her exact diagnosis remained uncertain. In view of this history, she underwent an antenatal echocardiogram, which showed borderline enlargement of the ascending aorta of 3.4 cm. Otherwise, the antenatal period was uneventful. At caesarean section, adhesions were noted, but the procedure was otherwise uncomplicated with an estimated total blood loss of 700 ml. At 21 hours postoperatively, she developed right iliac fossa pain and became profoundly hypotensive with a BP 45/35 mmHg and pulse rate 90 b.p.m. Physical examination revealed clammy cold peripheries and a distended abdomen. Her haemoglobin dropped from 10.9 g/dl preoperatively to 5.4 g/dl on an arterial blood gas sample taken urgently. She was immediately resuscitated with 5 units of red blood cells and 3 units of fresh frozen plasma. Bedside ultrasound scan showed minimal free fluid in the abdomen. Hence, a CT scan was urgently performed, which revealed an extensive retroperitoneal haematoma extending from the lower pole of the right kidney to the groin (Figure 1). The origin of the haematoma was thought to be an AVM arising off the anterior aorta just above the inferior mesenteric artery. It appeared to communicate with the anterior inferior vena cava just below the level of the renal veins. As the patient became clinically stable and given the significant risks of arterial puncture in a patient with a possible connective tissue disorder, angiography was not performed to further investigate or embolise the AVM. After consulting with the haematologist and vascular surgeons, it was decided to continue with conservative management. Due to the high risk of rebleed low-molecular weight heparin thromboprophylaxis was withheld. She remained clinically stable and was discharged home on postoperative day 6.

Discussion This case demonstrates a rare presentation of retroperitoneal haematoma, which was successfully managed conservatively. Prompt involvement of a multidisciplinary team, including vascular surgeons, haematologist and radiologist resulted in a favourable outcome. Various causes of retroperitoneal haematoma in obstetrics have been reported in the literature (Rafi and Muppala 2010), which include surgical trauma, rupture of abdominal or pelvic blood vessels, idiopathic, eclampsia/pre-eclampsia and prolonged labour. In our case, the origin of the haematoma was thought to be an AVM. A link between AVM rupture and pregnancy has been proposed but the majority of reports in the literature are related to intracranial AVMs and the risk of intracranial haemorrhage (Trivedi and Kirkpatrick 2003). This increase in rupture is thought to be caused by the increased cardiac output or circulatory effects of the elevated oestrogen levels. By acting on vasomotor tone, dilatation in both normal and abnormal vessels can occur (Elliot et al. 1985). AVMs therefore tend to dilate with the potential risk of rupture. In this case, the presence of a possible connective tissue disorder might have been associated with an increased risk of vascular fragility. The condition can lead to fatal haemorrhagic shock, therefore early recognition, active resuscitation and achievement of haemostasis are most important. Clinical manifestations of retroperitoneal haematomas include abdominal pain and hypovolaemic shock, as seen in our case. The immediate action to these signs and symptoms in many cases, would be an exploratory laparotomy, however in this case, it may not have revealed the source of bleeding. In our case, an exploratory laparotomy was not performed immediately because bedside ultrasound did not reveal any free fluid in the abdomen. Therefore, a retroperitoneal haematoma was suspected and confirmed with an urgent CT scan. Conservative management of retroperitoneal haematomas has been reported (Manabe et al. 2002; Gyimadu et al. 2010). Our patient responded well to resuscitation and remained stable and hence conservative management was continued. The haematoma itself might have served as a tamponade and achieved haemostasis. This case highlights the importance of incorporating the entire clinical picture and involving a multidisciplinary team when managing maternal collapse.

Prenatal diagnosis and molecular cytogenetic characterisation of a de novo 18p deletion.

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