Use of tissue samples in diagnosing diploid triploid mosaicism

Abstract

Diploid triploid mosaicism (DTM) is a rare genetic condition where there is an extra haploid set of chromosomes in mosaic form. We describe an infant for whom DTM was detected antenatally through amniocentesis. Prenatal counselling suggested a guarded prognosis. The infant’s phenotypic presentation and postnatal course reflect the varied presentation and prognosis associated with DTM. We highlight potential challenges in diagnosing DTM postnatally, with many having normal blood karyotype with 46 chromosomes.

Background

Diploid triploid mosaicism (DTM), also referred to as diploid/triploid mixoploidy, is an extremely rare condition where there is whole chromosome mosaicism. In affected individuals, some cells have a normal complement of 46 chromosomes (diploid), while other cells carry an extra copy of each chromosome, resulting in 69 chromosomes (triploid). DTM is distinct from triploidy, where all cells carry an extra set of haploid chromosomes, and is typically lethal in the first trimester of pregnancy.¹ Of individuals diagnosed with DTM, 75% have a normal blood karyotype showing typical diploid cells with 46 chromosomes.² Genetic testing of tissues other than blood is not routinely performed. As such, DTM is thought to be underdiagnosed as it is infrequently identified on routine genetic testing.²

There are approximately 45 cases of DTM reported in the literature to date.³ Clinical features are heterogeneous, with varying prognosis. A high incidence of spontaneous abortion or intrauterine death has been reported, with others surviving to adulthood.⁴ Information regarding prognosis and postnatal care is limited due to the small number of cases identified.

Case presentation

A healthy unrelated couple attended for routine prenatal care following natural conception. Severe intrauterine growth restriction was identified on ultrasound at 20 weeks’ gestation, with estimated fetal weight (EFW) less than the first centile. Fetal anatomy was otherwise normal. With parental consent, an amniocentesis was performed at 20 weeks’ gestation. A quantitative fluorescent (QF) PCR for trisomy 13, 18 and 21 and sex chromosome aneuploidy was performed. Karyotyping revealed mosaicism, with two cell lines identified on two independent amniocyte cultures. In six (20%) cells, a female complement of 69 chromosomes was identified, with three copies of all autosomes present. There was a normal diploid female complement in 24 (80%) cells, resulting in mos 69, XXX (6)/46, XX (24). QF PCR identified the origins of the triploidy cells as digenic.

Subsequent ultrasound scans at 24, 26, 28 and 31 weeks’ gestation indicated minimal interval growth, including biparietal diameter, abdominal circumference and femur length, with EFW persistently less than the first centile. Based on the limited literature available, the parents were counselled regarding the risk of fetal demise and the uncertainty regarding postnatal outcome, with palliative care being involved in postnatal care planning.

Following induction of labour, a female infant was delivered at 39+4 weeks’ gestation in good condition, with Apgar scores of 9 at 1 min and 9 at 5 min. Her birth weight was 2040 g (less than the first centile) and head circumference (HC) 32.5 cm (eighth centile). Clinical features included a small mouth, small chin, high-arched palate, single palmar creases, low-set ears and central hypotonia. Her cardiovascular, respiratory and abdominal exams were normal. She was discharged home on day 4 of life, taking all feeds per oral via syringe or teat as tolerated. Prior to discharge, the infant was referred to and accepted by community palliative care and hospice service.

Investigations

Peripheral blood karyotype and microarray were taken postnatally, revealing a normal 46, XX karyotype. A buccal smear was taken for interphase fluorescent in situ hybridisation analysis with chromosome 8 centromere (D8Z2) and chromosome 9 centromere (CEP9). A single copy gain involving both regions was detected in 38% of cells analysed, nuc ish (D8Z2, cen9)x3[38/100], consistent with DTM. Cranial, renal and hip ultrasonography were performed and were normal. A single small hepatic haemangioma was identified on ultrasound. At 7 months of age, the haemangioma had reduced in size. Histopathology assessment of the placenta was performed. The placenta was hypoplastic, weighing 272 g (less than the third centile), with normal-appearing parenchyma. There was a normal three-vessel umbilical cord, and the membranes were within normal limits.

Outcome and follow-up

On review at 6 weeks of age, the infant had good interval growth. She was teat feeding well and socially smiling. She had ongoing hypotonia, but otherwise examined well. At 4 months of age, her weight had more than doubled since birth (4290 g), tracking on the 0.01st centile, with HC on the 17th centile. She had generalised hypotonia, with all other system examinations within normal limits. At her 6-month developmental check, she continued to grow along her birth centiles, with weight on the 0.34th centile and HC on the 22nd centile, with persistent generalised hypotonia. At 15 months of age, she had persistent central hypotonia with hypermobility. She had delayed motor milestones and was unable to crawl or stand.

Despite her initial guarded prognosis, she remains well and her development is progressing on a stable trajectory. She is receiving input from physiotherapy and will receive ongoing monitoring of her growth and development. She has been discharged from community palliative care.

Discussion

This case highlights the heterogeneous nature of DTM, diagnostic considerations and challenges in counselling parents regarding clinical outcomes. DTM occurs in early embryogenesis, around the time of zygotic division, and features an extra set of chromosomes of either maternal or paternal origin. Compared with diploid cells, triploid cells appear to show restricted mosaicism and varied distribution, such that it is impossible to predict the lineage and anatomical pattern of the triploid cells and the clinical outcome.⁴

The varied distribution of triploid cells within tissues and the broad spectrum of clinical features and outcomes add to the challenge of establishing a diagnosis postnatally. Features identified in the reported case which are associated with DTM include growth retardation, hypotonia, microphthalmia, micrognathia and low-set ears. These are not pathognomonic of DTM.^{2 3}

Other reported features can include syndactyly (typically fingers 3 and 4) and ‘tree-frog like feet’, streaky skin pigmentation following Blaschko’s lines, clinodactyly, ventriculomegaly of the brain, clubfoot, congenital heart defects, body/facial asymmetry, cryptorchidism and premature menarche in female patients.^{2 4}

A single hepatic haemangioma was observed on abdominal ultrasound of the infant featured in our report. There were no cutaneous haemangiomas. Haemangiomas have not previously been described as a feature of DTM. Hepatic hamartomas have been reported in one infant who was being investigated for persistent hyperinsulinism. The infant was diagnosed with DTM when a triploid population of cells was detected in the cystic areas of the pancreas.⁵ This suggests abdominal ultrasound should be performed as part of the postnatal assessment of infants with DTM. At follow-up, we observed good interval growth and developmental progress. Significant catch-up growth has been reported to occur later in infancy, with some subsequently encountering issues with central obesity.³ Other potential issues presenting in childhood include intellectual disability, delayed walking, delayed speech and seizure disorders.^{2 6 7}

In the case presented, DTM was detected antenatally on amniocentesis. The method through which mosaicism is diagnosed antenatally should be taken into consideration. Antenatal genetic testing through either chorionic villus sampling (CVS) or amniocentesis may be considered where there is poor fetal growth or when congenital anomalies are seen on antenatal scans. At CVS testing, a sample of chorionic villus obtained from the placenta undergoes genetic testing, whereas amniocentesis facilitates genetic testing of the amniotic fluid, which includes cells from fetal skin, urine, as well as oral, nasal, tracheal and pulmonary fluids. An amniocentesis is therefore the best tissue to interrogate possible mosaic disorders.⁸ By contrast, a blood karyotype is performed on lymphocytes. Lymphocytes have a fast turnover in mitosis, and it is thought that aberrant cell lines are lost early on as rapid turnover prevents the necessary time for a haploid cell line to replicate. Thus, mosaicism may be missed on blood karyotyping as the cell line has disappeared from blood by the time of birth.

The literature reports that approximately 75% of patients with diploid triploid have normal blood karyotype and analysis of other tissues is required for a definitive diagnosis.² We found three cases reporting DTM diagnosis on karyotyping of peripheral blood sample, all of which had different clinical features and outcomes. For each of these cases, at least 30% of the cells displayed triploidy, demonstrating that the percentage of triploid cells likely impacts the likelihood of diagnosis through peripheral blood testing.^9–11

Diagnosing genetic conditions prenatally may impact on decisions to continue with a pregnancy and the care of the infant after birth. Prenatally diagnosed DTM and the associated outcomes are infrequently reported. Providing accurate prognostic information to families antenatally is challenging. A liveborn infant with prenatally diagnosed DTM on amniocentesis was first described in 2012.¹² In their report, the authors described a liveborn female infant with some similar facial features to the infant outlined in our case report. They report delayed attainment in developmental milestones, including gross motor, fine motor and language at 16 months of age.¹² Wegner et al¹³ reported a case of DTM diagnosed at 23 weeks’ gestation by amniocentesis for investigation of polyhydramnios and cardiac anomaly. This pregnancy ended in intrauterine fetal death at 25 weeks’ gestation.¹³ DTM diagnosed on CVS has been associated with variable outcomes.^{4 14} Carson et al⁴ reported on a series of eight cases where DTM was detected on CVS culture. Both CVS and amniocentesis were performed in two cases. In their first case, there were no triploid cells present in the amniotic fluid and was associated with a normal-appearing male infant. In the second case, they described a fetus with multiple anomalies and a high proportion of triploid cells on both CVS and amniocentesis, which were associated with intrauterine fetal demise (IUFD). Of the remaining cases, four were associated with IUFD, one termination of pregnancy and one neonatal death following extreme preterm birth.⁴

Although DTM detected on amniocentesis more likely suggests fetal involvement,^{4 13} one case of DTM has been reported in a child with normal amniocentesis. The child was subsequently diagnosed at 1 year of age on culture of skin fibroblasts.¹⁵ This highlights the importance of postnatal investigation. The infant featured in our report returned a normal peripheral blood karyotype and microarray. The result of mosaicism on amniocentesis assisted in directing postnatal investigations. A buccal smear was performed, which confirmed DTM. DTM is a condition that is often forgotten due to its apparently low incidence. There have been reported cases where a genetic condition was suspected after birth; however, a diagnosis of DTM was delayed by months or years as it was not detected on peripheral blood karyotyping.^{6 14} Delayed diagnosis can impact on provision of healthcare and access to appropriate auxiliary services. It also prevents accurate genetic counselling. The recurrence risk of DTM is the same as in the general population; however, without the diagnosis, the parents may be given higher empiric recurrence risks (ie, 3%–5%), which may impact future family planning. When looking to establishing a genetic diagnosis for infants with normal peripheral blood karyotype, it is important to consider mosaicism and proceed with testing of alternative sites, such as the buccal mucosa, skin or urine.^{2 3 6}

The limited knowledge available on DTM impacts on how we counsel families with an affected fetus regarding the expected outcomes and the clinical management and investigation. The case presented highlights these challenges. These parents met with paediatric palliative care antenatally as it was expected that their infant would have a short life. Contrary to expectation, the infant was well at birth and is currently thriving. Survival may not relate to the proportion of triploid cells on culture,² but to the degree to which triploidy is expressed in the tissues, in particular the brain.¹² Although there is an apparently high incidence of fetal demise associated with DTM,⁴ liveborn infants appear to survive long term with varying degrees of intellectual disability.^{2 3 12} The parental origin of the additional chromosome may also influence the outcome. It has been proposed that infants whose triploid cells are of maternal origin may have a better prognosis than those whose triploid cells are paternal.²

Learning points.

• Diploid triploid mosaicism (DTM) is difficult to diagnose as it is often not detected on routine testing.

• Amniocentesis is the ideal tissue for identifying mosaicism; however, postnatal confirmation is advised.

• DTM is being underdiagnosed as it is infrequently detectable in the blood; therefore, genetic testing of alternative tissues should be considered where a phenotype is suggestive of a mosaic disorder.

• DTM should be considered when significant intrauterine growth restriction is seen on prenatal scans.

• Patients with DTM have extremely variable outcomes, making antenatal counselling difficult.

Ethics statements

Patient consent for publication

Parental/guardian consent obtained.