Expanding the understanding of telomere biology disorder with reports from two families harboring variants in ZCCHC8 and TERC.

Telomere biology disorder (TBD) can present within a wide spectrum of symptoms ranging from severe congenital malformations to isolated organ dysfunction in adulthood. Diagnosing TBD can be challenging given the substantial variation in symptoms and age of onset across generations. In this report, we present two families, one with a pathogenic variant in ZCCHC8 and another with a novel variant in TERC. In the literature, only one family has previously been reported with a ZCCHC8 variant and TBD symptoms. This family had multiple occurrences of pulmonary fibrosis and one case of bone marrow failure. In this paper, we present a second family with the same ZCCHC8 variant (p.Pro186Leu) and symptoms of TBD including pulmonary fibrosis, hematological disease, and elevated liver enzymes. The suspicion of TBD was confirmed with the measurement of short telomeres in the proband. In another family, we report a novel likely pathogenic variant in TERC. Our comprehensive description encompasses hematological manifestations, as well as pulmonary and hepatic fibrosis. Notably, there are no other reports which associate this variant to disease. The families expand our understanding of the clinical implications and genetic causes of TBD.

Microvillus Inclusion Disease Caused by MYO5B: Different Presentation and Phenotypes Despite Same Mutation.

Microvillus inclusion disease (MVID) is associated with specific variants in the MYO5B gene causing disrupt epithelial cell polarity. MVID may present at birth with intestinal symptoms or with extraintestinal symptoms later in childhood. We present 3 patients, of whom 2 are siblings, with MYO5B variants and different clinical manifestations, ranging from isolated intestinal disease to intestinal disease combined with cholestatic liver disease, predominant cholestatic liver disease clinically similar to low-gamma-glutamyl transferase PFIC, seizures, and fractures. We identified 1 previously unreported MYO5B variant and 2 known pathogenic variants and discuss genotype-phenotype correlations of these variants. We conclude that MVID may present phenotypically different and mimic other severe diseases. We suggest that genetic testing is included early during diagnostic investigations of children with gastrointestinal and cholestatic presentation.

Toxoplasma gondii-associated Placentitis in the absence of maternal seroconversion.

Severe granulomatous chronic villitis with focal remnants of Toxoplasma was confirmed by immunohistochemistry and DNA-based methods in the placenta from a child that died four days after birth. The immunocompetent mother was seronegative for Toxoplasma at delivery and 10 months later. Placental infection may happen without maternal systemic infection.

Conjoined twins after single blastocyst transfer: a case report including detailed time-lapse recording of the earliest embryogenesis, from zygote to expanded blastocyst.

Conjoined twins are estimated to occur in 1:50 000 pregnancies. Eighteen cases of pregnancies achieved by ART have been published of which three were achieved after single embryo transfer, allowing discussion of embryo characteristics. We report, to the best of our knowledge, the first case of parapagus conjoined twins after ART. Furthermore, this is the first report of conjoined twins with detailed morphokinetics of the earliest embryogenesis from zygote to expanded and hatched blastocyst stage. The case zygote had three refractile bodies, which were all allocated to one blastomere at first cleavage following an asynchronous pronuclei fading. Within 2 h, this blastomere cleaved to four and fragmented. The remaining blastomere cleaved symmetrically and regularly and a blastocyst (score: 4AB) was vitrified 120 h after IVF. Pregnancy was achieved following a frozen-thawed single blastocyst transfer. The etiopathogenetic mechanism of the origin of conjoined twins is unknown and several hypotheses exist. The morphokinetics in the present case and morphology of other reported cases will be discussed in this context.

A common deletion in the growth hormone receptor gene (d3-GHR) in the offspring is related to maternal placental GH levels during pregnancy.

BACKGROUND: A common growth hormone receptor polymorphism with deletion of exon 3 (d3-GHR) has previously been linked to increased postnatal growth on the one hand and decreased fetal growth on the other. Regulation of fetal growth is positively dependent on secretion of placental GH (hGH-V). OBJECTIVE: We explored the effect of the fetal d3-GHR genotype on maternal serum levels of hGH-V and fetal growth. The cellular localization of hGH-V synthesis and the GH receptors were determined in normal placentas. METHODS: 43 healthy mother-child pairs were examined during pregnancy with measurements of hGH-V during third trimester, and serial ultrasound measurements determined fetal growth rate. Birth anthropometrics were obtained. The GHR genotype of the child was analysed postnatally. Immunohistochemical (IHC) analysis was conducted on four placentas. RESULTS: The presence of the d3-GHR genotype was associated with a markedly reduced concentration of hGH-V in maternal serum (ß -0.52, SE 0.24, p = 0.04) compared to those who had a fl/fl genotype. Accordingly, a tendency towards reduced fetal growth rate during third trimester (ß -25.8, SE 12.7, p = 0.05) and a lower birth weight were found among carriers of the d3-GHR allele, but these associations did not reach statistical significance (p = 0.08). IHC analysis showed expression of placental GH and GHR in the villous syncytiotrophoblast, the extravillous trophoblast, and the decidual cells and smooth muscle cells in chorionic vessels. CONCLUSIONS: The presence of the d3-GHR polymorphism in the fetus was associated with lower maternal serum levels of hGH-V, decreased fetal growth rate in third trimester and lower birth weight compared to the wildtype.

Apheresis principles in a patient with chronic myeloid leukemia during pregnancy: challenges in cell separation and assessing transcript levels.

BACKGROUND: Chronic myeloid leukemia (CML) is rarely diagnosed in pregnant women. CASE REPORT: We report a case of a pregnant woman who presented with a leukocyte count of 250 × 109 cells/L at gestational age (GA) 26 weeks and was diagnosed with CML in the chronic phase. Because the patient deliberately opted out of interferon α and tyrosine kinase inhibitor treatment, the main goal was to reduce the leukocyte count to postpone delivery beyond the number of weeks considered severely premature and avoid thromboembolic complications while continuously evaluating the clinical safety of the mother and fetus. Hence therapeutic leukapheresis was initiated, and we report the first application of an apheresis approach for this procedure using the Spectra Optia instrument without sedimentation agents. Leukapheresis was conducted 2 to 4 times per week for 9 weeks. RESULTS: During treatment the leukocyte count decreased remarkably, and the patient developed lymphopenia together with a paradoxical increase in her blood platelet count. Premature labor was induced at GA 35 weeks, and a healthy boy was delivered. Thereafter, the patient initiated imatinib treatment and was in major molecular and complete cytogenetic remission after 1 year. Despite the remarkable reduction of the leukocyte count, we observed a pronounced increase in expression of BCR-ABL1 transcripts, implying the need for close monitoring of patients with CML during pregnancy. CONCLUSIONS: We report a pregnant woman who was diagnosed with CML and treated solely with apheresis procedures using the Spectra Optia instrument for 9 weeks, ensuring the safe delivery of her child.

Spontaneous Septostomy in a Twin Pregnancy Causing Fatal Amniotic Band Syndrome.

Complications due to spontaneous septostomy of the dividing membrane in monochorionic diamniotic pregnancies are rarely described. Herein, we report the case of a preterm female neonate from a monochorionic diamniotic twin pregnancy delivered by caesarean section at 32 weeks of gestation. She was born with a broad band of a transparent membrane-like material firmly attached to her lower abdomen. Postnatally, she developed respiratory distress syndrome and persistent pulmonary hypertension, complicated by bilateral pneumothorax. She died due to respiratory failure when she was 1 day old. Her twin sister survived with no malformations. At postmortem examination, the neonate had severe lung hypoplasia, and the attached material was diagnosed as the dividing septum. We hypothesize that the lung hypoplasia was secondary to local oligohydramnios, which developed as a consequence of the twin being firmly stuck in the defect of the dividing membrane. To our best knowledge, spontaneous septostomy causing an ultimately fatal amniotic band syndrome has not previously been described.

Rare novel variants in the ZIC3 gene cause X-linked heterotaxy.

Variants in the ZIC3 gene are rare, but have demonstrated their profound clinical significance in X-linked heterotaxy, affecting in particular male patients with abnormal arrangement of thoracic and visceral organs. Several reports have shown relevance of ZIC3 gene variants in both familial and sporadic cases and with a predominance of mutations detected in zinc-finger domains. No studies so far have assessed the functional consequences of ZIC3 variants in an in vivo model organism. A study population of 348 patients collected over more than 10 years with a large variety of congenital heart disease including heterotaxy was screened for variants in the ZIC3 gene. Functional effects of three variants were assessed both in vitro and in vivo in the zebrafish. We identified six novel pathogenic variants (1,7%), all in either male patients with heterotaxy (n=5) or a female patient with multiple male deaths due to heterotaxy in the family (n=1). All variants were located within the zinc-finger domains or leading to a truncation before these domains. Truncating variants showed abnormal trafficking of mutated ZIC3 proteins, whereas the missense variant showed normal trafficking. Overexpression of wild-type and mutated ZIC protein in zebrafish showed full non-functionality of the two frame-shift variants and partial activity of the missense variant compared with wild-type, further underscoring the pathogenic character of these variants. Concluding, we greatly expanded the number of causative variants in ZIC3 and delineated the functional effects of three variants using in vitro and in vivo model systems.

The outcome of antenatal ultrasound diagnosed anomalies of the kidney and urinary tract in a large Danish birth cohort.

OBJECTIVE: Antenatal ultrasound diagnosed anomalies of the kidney and urinary tract (AUDAKUT) are reported in 0.3%-5% on prenatal ultrasound (US) and 0.3%-4.5% on postnatal US. The anterior-posterior diameter of the renal pelvis (APD) is an essential measurement. Series with low threshold values of APD prenatally and postnatally will include healthy infants. It is important to avoid follow-up of such infants. INTERVENTIONS: In 2006, new Danish guidelines for AUDAKUT were introduced. AIM OF STUDY: Investigations of incidences and type of AUDAKUT based on Danish guidelines, including long-term follow-up. DESIGN: Cohort study. SETTING: Copenhagen University Hospital Hvidovre and Copenhagen University Hospital Rigshospitalet, Denmark. PATIENTS: Consecutive cases with AUDAKUT in the second and third trimesters, which were either terminated before 22 completed weeks of gestation or born in the 8-year period January 2006-December 2013. Patients were followed until June 2014. RESULTS: 50 193 live born children and 24 terminated fetuses (0.05%) were included. The prevalence of AUDAKUT was only 0.39% prenatally, 0.29% at first postnatal US and 0.22% at the end of follow-up, including terminated cases. The greater the prenatal and postnatal APD, the higher risk of febrile urinary tract infection (fUTI) and surgical intervention, and lower probability of resolution. 25% of the identified patients had fUTI and/or surgery. CONCLUSIONS: We recommend threshold values of APD at least 10 mm in the third trimester and in general at least 12 mm at first postnatal US for intensive follow-up. In this largest to date unselected birth cohort of AUDAKUT, the incidences of clinically significant AUDAKUT were in the lowest range of those previously published.

Gestational trophoblastic diseases - clinical guidelines for diagnosis, treatment, follow-up, and counselling.

Hydatidiform mole is treated with surgical uterine evacuation with suction and blunt curettage (D). Medical uterine evacuation should not be used (C). On clinical suspicion of hydatidiform mole, one representative sample of the evacuated tissue is fixed for histopathologic investigation and one is forwarded unfixed for genetic analysis (D). Serum hCG is measured on suspicion of hydatidiform mole. At the time of the uterine evacuation, the initial hCG is measured (A). After a hydatidiform mole that is both triploid and partial, serum hCG is measured weekly until there are two consecutive undetectable values (< 1 or < 2), after which the patient can be discharged from follow-up (C). After a diploid hydatidiform mole, a complete mole, or a hydatidiform mole without valid ploidy determination, serum hCG is measured weekly until the value is undetectable (< 1 or < 2). If serum hCG is undetectable within 56 days after evacuation, the patient can be discharged from follow-up after an additional four monthly measurements. If serum hCG is first normalised after 56 days, the patient is follow-up with monthly serum hCG measurement for six months. Safe contraception should be used during the follow-up period (A). If hCG stagnates (less than 10% fall over three measurements), increases, or if hCG can be demonstrated for longer than 6 months, the patient by definition has persistent trophoblastic disease (PTD). A chest X-ray should be taken and a gynaecologic ultrasound scanning performed. The patient is referred to oncologic treatment (A). Uterine re-evacuation as a treatment for PTD can, in general, not be recommended because the rate of remission is low, and there is the risk of perforation of the uterus (C). In all following pregnancies, the woman is offered an early ultrasound scan, e.g. in gestational week eight (D). Eight weeks after termination of all future pregnancies, serum hCG is measured (D). In PTD and invasive hydatidiform mole, the primary treatment is MTX, either orally every third week or IV every week (B). In MTX-resistant PTD, IV act D is added (or replaces the MTX) (B). Third line chemotherapy is BEP or EP, alternatively EMA-CO (B). Choriocarcinoma is primarily treated with chemotherapy. Hysterectomy and/or resection of metastases are possible treatments (A). Placental site trophoblastic tumour (PSTT) and epithelioid trophoblastic tumour (ETT) are primarily treated with hysterectomy. In the case of disseminated disease, chemotherapy is considered (A). The risk of reoccurrence after trophoblastic disease treated with chemotherapy is approximately 3%. Most reoccurrences are seen within 12 months, and for this reason monitoring of hCG is recommended for one year, the first third months once or twice a month, thereafter every second to third month. Patients with PSTT and ETT are monitored with measurement of hCG throughout their lifetimes (C). In genetically verified twin pregnancy with hydatidiform mole and a living foetus, the pregnancy can continue if serum hCG is monitored and ultrasound scans regularly performed, and possible obstetric complications dealt with (C). In the case of recurrent hydatidiform mole and/or familial hydatidiform mole, patients should be referred to genetic workup and counselling (C). Women with a hereditary disposition to hydatidiform mole because of a mutation in NLRP7 should be informed of the possibility of becoming pregnant via egg donation (C).

Kinetics of silica nanoparticles in the human placenta.

The potential medical applications of nanoparticles (NPs) warrant their investigation in terms of biodistribution and safety during pregnancy. The transport of silica NPs across the placenta was investigated using two models of maternal-foetal transfer in human placenta, namely, the BeWo b30 choriocarcinoma cell line and the ex vivo perfused human placenta. Nanotoxicity in BeWo cells was examined by the MTT assay which demonstrated decreased cell viability at concentrations >100 µg/mL. In the placental perfusion experiments, antipyrine crossed the placenta rapidly, with a foetal:maternal ratio of 0.97 ± 0.10 after 2 h. In contrast, the percentage of silica NPs reaching the foetal perfusate after 6 h was limited to 4.2 ± 4.9% and 4.6 ± 2.4% for 25 and 50 nm NPs, respectively. The transport of silica NPs across the BeWo cells was also limited, with an apparent permeability of only 1.54 × 10(-6) ± 1.56 × 10(-6) cm/s. Using confocal microscopy, there was visual confirmation of particle accumulation in both BeWo cells and in perfused placental tissue. Despite the low transfer of silica NPs to the foetal compartment, questions regarding biocompatibility could limit the application of unmodified silica NPs in biomedical imaging or therapy.

Prenatal detection of congenital heart disease in a low risk population undergoing first and second trimester screening.

OBJECTIVES: The prenatal detection rate of congenital heart disease (CHD) is low compared with other fetal malformations. Our aim was to evaluate the prenatal detection of CHD in Eastern Denmark. METHODS: Fetuses and infants diagnosed with CHD in the period 01.01.2008-31.12.2010 were assessed regarding prenatal detection rate and accuracy, as well as correlation with nuchal translucency (NT) thickness. RESULTS: Out of 86 121 infants, 831 were born with CHD (0.96%). The prenatal detection rate of 'all CHD' was 21.3%, of 'Major CHD' 47.4%. Full agreement between prenatal and postnatal/autopsy findings was found in 96% of prenatally detected diagnoses. An NT thickness >95(th) percentile was found in 15.0% fetuses with 'Major CHD'. Of 'Major CHDs' detected prenatally, 77% were picked up at the time of the malformation scan at weeks 18-21. CONCLUSIONS: Nearly half of 'Major CHDs' were detected prenatally. The prenatal cardiac diagnoses showed a high degree of accuracy. Increased NT thickness as a screening tool for CHD performed moderately but is an important high risk group for specialist examination. A minority of the prenatally detected CHDs was identified because of extra scans performed in high risk pregnancies. © 2014 John Wiley & Sons, Ltd.

Pathologic evaluation of normal and perfused term placental tissue.

This study reports for the 1st time the incidence and interobserver variation of morphologic findings in a series of 34 term placentas from pregnancies with normal outcome used for perfusion studies. Histologic evaluation of placental tissue is challenging, especially when it comes to defining "normal tissue" versus "pathologic lesions." A scoring system for registration of abnormal morphologic findings was developed. Light microscopic examination was performed independently by 2 pathologists, and interobserver variation was analyzed. Findings in normal and perfused tissue were compared and selected findings were tested against success parameters from the perfusions. Finally, the criteria for frequent lesions with fair to poor interobserver variation in the nonperfused tissue were revised and reanalyzed. In the perfused tissue, the perfusion artefact "trophoblastic vacuolization," which is believed to represent dilated transtrophoblastic channels, was reproducible and significantly correlated to the perfusion marker "fetal leakage." In longer perfusions, microscopy of the perfused cotyledon revealed bacteria in the fetal vessels. This finding led to an adjustment in the perfusion protocol with addition of antibiotics to the medium. In the "normal" tissue, certain lesions were very frequent and showed only fair or poor interobserver agreement. Revised minimum criteria for these lesions were defined and found reproducible. This study has emphasized the value of pathologic examination as a supplement in placental perfusion models. Examination of the perfused cotyledon for trophoblastic vacuolization is recommended as an additional quality marker in perfusion models. The study also underlines the need for exact definitions of abnormality in frequent placental lesions.

Ghrelin in the fetal pancreas - a digital quantitation study.

Ghrelin is a hormone produced by specialized neuroendocrine cells located in the fetal pancreas. In the adult, ghrelin has multiple effects, but in the fetus the role of ghrelin and the distribution of ghrelin-producing cells is not well documented. The aim of this study was to describe and quantitate the number of ghrelin positive cells in the pancreas during gestation. The material consisted of pancreatic tissue from 19 fetuses at different gestational ages. Immunohistochemical staining was performed, and the expression was quantitated using an automated digital image analysis system. The results showed ghrelin-producing cells as scattered single cells in ductular structures and acini throughout the gestation. From midgestation they were also found in the periphery of the islets as a rim of cells. A tendency towards a high ghrelin expression during early gestation and a stable expression from midgestation to term was observed. In conclusion, the effects of fetal ghrelin are not fully understood, but the varying distribution of ghrelin positive cells indicates different effects of ghrelin during development.

Correlation between prenatal diagnosis by ultrasound and fetal autopsy findings in second-trimester abortions.

We evaluated the correlation between prenatal diagnosis by ultrasound and autopsy findings, based on 52 second-trimester pregnancies terminated due to fetal malformations or chromosome aberrations diagnosed at a gestational age of 12-25 weeks. In 24 pregnancies, there was full agreement between ultrasound and autopsy. In 23 fetuses, the main diagnosis was confirmed and additional or more specific findings were observed on autopsy. In five fetuses, there were considerable differences. Discrepancies between ultrasound and autopsy findings were mainly anomalies undetectable by ultrasound and thus expected; however, about one-third of the discrepancies were not expected, representing findings that were 'missed' at ultrasound. The main ultrasound diagnoses were confirmed in the majority of the pregnancies, but the additional information obtained at autopsy in more than half of the fetuses clearly shows the value and benefit of postmortem fetal examination following termination of a pregnancy.

[Increased nuchal translucency in osteogenesis imperfecta]. / Forstørret nakkefold kan ses ved osteogenesis imperfecta.

A limited number of reports published since 2001 have described an association between increased nuchal translucency (NT) and osteogenesis imperfecta (OI). We report a new case which underlines the frequency of this association as well as the importance of follow-up and genetic evaluation. In the present case, ultrasound scanning at 13 weeks of gestation showed a NT of 3.2 mm and no other pathological findings. At 20 weeks a severe skeletal dysplasia was diagnosed by ultrasound. The pathology report of the aborted foetus indicated OI, and DNA analysis confirmed a COL1A1 mutation.

MMSET is highly expressed and associated with aggressiveness in neuroblastoma.

MMSET (WHSC1/NSD2) is a SET domain-containing histone lysine methyltransferase the expression of which is deregulated in a subgroup of multiple myelomas with the t(4;14)(p16;q32) translocation associated with poor prognosis. Recent studies have shown that MMSET mRNA levels are increased in other tumor types as well. We have carried out immunohistochemical staining of tissue microarrays and found that MMSET protein is frequently and highly expressed in neuroblastoma (MMSET positive in 75% of neuroblastomas, n = 164). The expression level of MMSET in neuroblastomas was significantly associated with poor survival, negative prognostic factors, and metastatic disease. Moreover, a subset of neuroblastomas for which pre- and postchemotherapy biopsies were available displayed a strong decrease in MMSET protein levels after chemotherapy. In agreement with neuroblastomas becoming more differentiated after treatment, we show that retinoic acid-induced differentiation of human neuroblastoma cells in vitro also leads to a strong decrease in MMSET levels. Furthermore, we show that the high levels of MMSET in normal neural progenitor cells are strongly downregulated during differentiation. Importantly, we show that MMSET is required for proliferation of neuroblastoma cells and brain-derived neural stem cells. Taken together, our results suggest that MMSET is implicated in neuroblastomagenesis possibly by supporting proliferation of progenitor cells and negatively regulating their differentiation. In this respect, MMSET might be a strong candidate therapeutic target in a subset of neuroblastomas with unfavorable prognosis.

Massive fetomaternal hemorrhage caused by an intraplacental choriocarcinoma: a case report.

Background. Intraplacental choriocarcinoma is a rare but highly malignant trophoblastic neoplasm. When found near term the risk of maternal metastasis is high because of the late diagnosis. Case. We describe a case of an intraplacental choriocarcinoma diagnosed postpartum after a near-term delivery of a severely anemic infant. A fetomaternal hemorrhage resulted in a hemoglobin concentration in the infant of only 2,1 g/dL. Neither mother nor child showed signs of metastatic disease. The macroscopic examination showed a hydropic placenta weighing more than 1 kilogram. Microscopy showed an intraplacental choriocarcinoma 3 cm in diameter. The tumor had infiltrated the maternal basal plate. Conclusion. Fetomaternal bleeding is a rare form of presentation of choriocarcinoma but its presence should always warrant detailed examination of placenta, mother, and infant.

Quality assessment of a placental perfusion protocol.

Validation of in vitro test systems using the modular approach with steps addressing reliability and relevance is an important aim when developing in vitro tests in e.g. reproductive toxicology. The ex vivo human placental perfusion system may be used for such validation, here presenting the placental perfusion model in Copenhagen including control substances. The positive control substance antipyrine shows no difference in transport regardless of perfusion media used or of terms of delivery (n=59, p<0.05). Negative control studies with FITC marked dextran correspond with leakage criteria (<3 ml h(-1) from the fetal reservoir) when adding 2 (n=7) and 20mg (n=9) FITC-dextran/100 ml fetal perfusion media. Success rate of the Copenhagen placental perfusions is provided in this study, including considerations and quality control parameters. Three checkpoints suggested to determine success rate revealed that 15% of the cannulated placentae received in one year (n=202) were successfully perfused.