Universal chromosomal microarray analysis reveals high proportion of copy-number variants in low-risk pregnancies.

OBJECTIVES: To evaluate the yield and utility of the routine use of chromosomal microarray analysis (CMA) for prenatal genetic diagnosis in a large cohort of pregnancies with normal ultrasound (US) at the time of genetic testing, compared with pregnancies with abnormal US findings. METHODS: We reviewed all prenatal CMA results in our center between November 2013 and December 2018. The prevalence of different CMA results in pregnancies with normal US at the time of genetic testing ('low-risk pregnancies'), was compared with that in pregnancies with abnormal US findings ('high-risk pregnancies'). Medical records were searched in order to evaluate subsequent US follow-up and the outcome of pregnancies with a clinically relevant copy-number variant (CNV), i.e. a pathogenic or likely pathogenic CNV or a susceptibility locus for disease with > 10% penetrance, related to early-onset disease in the low-risk group. RESULTS: In a cohort of 6431 low-risk pregnancies that underwent CMA, the prevalence of a clinically significant CNV related to early-onset disease was 1.1% (72/6431), which was significantly lower than the prevalence in high-risk pregnancies (4.9% (65/1326)). Of the low-risk pregnancies, 0.4% (27/6431) had a pathogenic or likely pathogenic CNV, and another 0.7% (45/6431) had a susceptibility locus with more than 10% penetrance. Follow-up of the low-risk pregnancies with a clinically significant early-onset CNV revealed that 31.9% (23/72) were terminated, while outcome data were missing in 26.4% (19/72). In 16.7% (12/72) of low-risk pregnancies, an US abnormality was discovered later on in gestation, after genetic testing had been performed. CONCLUSION: Although the background risk of identifying a clinically significant early-onset abnormal CMA result in pregnancies with a low a-priori risk is lower than that observed in high-risk pregnancies, the risk is substantial and should be conveyed to all pregnant women. © 2020 International Society of Ultrasound in Obstetrics and Gynecology.

Single-nucleotide polymorphism-based chromosomal microarray analysis provides clues and insights into disease mechanisms.

OBJECTIVE: Chromosomal microarray analysis (CMA) is the modality of choice for prenatal diagnosis in pregnancy with fetal malformation, as it has a high diagnostic yield for microdeletion/duplication syndromes. The aim of this study was to demonstrate the additional utility of single-nucleotide polymorphism (SNP)-based CMA in diagnosing monogenic diseases, imprinting disorders and uniparental disomy (UPD). METHODS: CMA was performed using Affymetrix CytoScan array, for all indications in 6995 pregnancies, at a tertiary referral hospital from November 2013 to June 2018. We describe four cases that had a CMA result that provided a more comprehensive understanding of the complex genetic mechanisms underlying the clinical presentation. RESULTS: In the first fetus, CMA was performed due to intrauterine growth restriction and revealed a 75 kbp maternally inherited microdeletion encompassing the Bloom syndrome gene (BLM). A diagnosis of Bloom syndrome was made upon identifying a paternally inherited common Ashkenazi founder mutation. In the second case, CMA was performed due to severely abnormal maternal serum analytes and revealed a deletion in 14q32.2q32.31 on the maternally inherited copy, leading to a diagnosis of Kagami-Ogata syndrome, which is an imprinting disorder. In the third case, amniocentesis was performed because of late-onset fetal macrosomia and mild polyhydramnios. CMA detected a deletion encompassing the locus of Prader-Willi/Angelman syndrome. In the fourth case, amniocentesis was performed due to maternal cytomegalovirus seroconversion. Maternal UPD of the entire long arm of chromosome 11 was detected. CONCLUSION: Prenatal CMA, based on oligo and SNP platforms, increases the diagnostic yield and enables a wider spectrum of disorders to be detected through the identification of complex genetic etiologies beyond only copy number variants. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

Fetal exome sequencing: yield and limitations in a tertiary referral center.

OBJECTIVE: To explore the indications for and diagnostic outcomes of fetal exome sequencing in a tertiary referral center. METHODS: Between 2012 and 2017, 77 unrelated fetal samples from pregnancies referred to our center underwent exome sequencing. The cohort included 37 fetuses, 36 products of conception (from cases of pregnancy termination or intrauterine fetal death), one case with DNA from both the fetus and a previous termination of pregnancy, and three cases with DNA of unknown origin. Exome sequencing was performed on DNA extracted from amniocytes or fetal tissue and, in some cases, from parental peripheral blood. Indications, turnaround time, diagnostic rates and pregnancy outcomes were investigated. Diagnostic yield was analyzed according to consanguinity (yes or no), sample type (proband only, or trio or other) and referral indication (malformation or isolated nuchal translucency (NT)). RESULTS: The most common indication for fetal exome sequencing was multiple malformations (21/77, 27%), followed by isolated brain malformation (15/77, 19%). Twelve (16%) fetuses were referred for isolated increased NT. Exome analysis was diagnostic for 16 fetuses (21%); when subclassified into fetal malformations vs isolated increased NT it became clear that exome analysis did not reveal any known or probable pathogenic variants in cases referred for isolated increased NT, whereas, among the remaining fetuses, a molecular diagnosis was reached in 16/65 (25%). Proband-only cases received a diagnosis more often than did cases that had trio exome sequencing. CONCLUSIONS: Exome sequencing has the potential to provide molecular diagnoses in cases in which conventional prenatal cytogenetic testing is negative. Referral bias of consanguineous cases could account for the high diagnostic rate of proband-only sequencing. Syndrome-specific prognostic information enables parents to make informed decisions, whereas challenges include time limitations and variant interpretation in the setting of non-specific fetal findings. As we report only established disease-gene associations, further segregation and functional studies in a research setting are expected to increase significantly the diagnostic yield. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Role of late amniocentesis in the era of modern genomic technologies.

OBJECTIVE: Traditionally, amniocentesis is performed between 17 and 23 weeks of gestation. This enables decisions regarding the course of pregnancy to be made before viability. Less frequently, amniocentesis is performed in the third trimester. Advanced genomic technologies such as chromosomal microarray analysis (CMA) provide more detailed information about the fetus compared with traditional G-banded chromosomal analysis. The aim of this study was to assess the indications for and safety of late amniocentesis, genetic-test results (especially in the context of CMA technology) and outcome of pregnancies that underwent the procedure after 24 weeks. METHODS: Medical records were analyzed retrospectively of all women in whom amniocentesis was performed at a gestational age of 24 + 0 to 38 + 6 weeks, at Hadassah Medical Center, between June 2013 and March 2017. Parameters investigated included indications for late amniocentesis, complications, CMA results and pregnancy outcome. RESULTS: During the study period, 291 women (303 fetuses, 277 singleton and 14 twin pregnancies; in two twin pairs, one fetus was terminated before amniocentesis) underwent late amniocentesis. CMA was performed in all instances of amniocentesis. The most frequent indication was abnormal sonographic finding(s) (204/303 fetuses, 67%). Preterm delivery occurred in 1.7% and 5.1% of pregnancies within the first week and within 1 month following the procedure, respectively. Aneuploidy was detected in nine (3%) fetuses and nine (3%) others had a pathogenic/likely pathogenic copy number variant, suggesting that CMA doubled the diagnostic yield of traditional karyotyping. Maximal diagnostic yield (17.5%) was achieved for the subgroup of fetuses referred with abnormal sonographic findings in two or more fetal anatomical systems. Variants of uncertain significance or susceptibility loci were found in another nine (3%) fetuses. CONCLUSIONS: In pregnancies undergoing late amniocentesis, CMA increased detection rates of fetal abnormalities and had a shorter turnaround time compared with traditional chromosomal analysis; therefore, late amniocentesis may serve as a helpful tool for detecting fetal abnormalities or reassuring parents following late-appearing abnormal sonographic findings. However, CMA may expose findings of uncertain significance, about which the couple should be precounseled. The procedure appears to be safe. Copyright © 2018 ISUOG. Published by John Wiley & Sons Ltd.

Congenital myopathy due to myosin heavy chain 2 mutation presenting as chronic aspiration pneumonia in infancy.

A 7-week-old infant presented with persistent noisy breathing and aspirations during swallowing. Neurological examination and brain MRI were normal. His 12-year-old brother underwent pneumonectomy at the age of 10 years due to recurrent aspirations leading to severe lung damage. The older brother developed subsequently ophthalmoplegia and nystagmus along with mild weakness of the neck flexors and proximal muscles. Exome analysis revealed homozygosity for a novel truncating mutation p.G800fs27* in the Myosin Heavy Chain 2 (MYH2) gene in both brothers, while parents and an unaffected sibling were heterozygous. A muscle biopsy from the older brother showed absence of type-2 muscle fibers and predominance of type-1 fibers. The aspirations causing pneumonia likely result from weakness of the laryngeal muscles, normally rich in type-2 fibers. The findings expand the phenotypic spectrum of MYH2 deficiency. MYH2 mutations should be included in the differential diagnosis of infants presenting with recurrent aspirations.

Group A streptococcus: is there a genital carrier state in women following infection?

Group A streptococcus (GAS) is a rare but serious cause of postpartum and gynecological infections. There are no follow-up or prophylaxis guidelines for women with previous GAS genital infection. We aimed to evaluate the incidence of long-term gynecological carrier state in patients with a history of genital GAS infection. This is a prospective study of women who had a genital GAS infection and were followed for 1 year from the date of isolation. Cultures were obtained every 3-4 months. As a control group, women with no previously documented GAS infection were screened for GAS. Twenty-five women with a previous GAS infection participated in the study. Two of the 25 patients had positive vaginal GAS cultures during follow-up, giving a carrier rate of 8 %. Four hundred and thirty-six women participated in the control group; none was a carrier of GAS (p < 0.003). We found that common gynecological procedures were occasionally associated with invasive GAS infection. A significant rate of carriers was found among women with previous GAS genital infection. Common office procedures can be related to severe GAS infection. Consideration should be given to screening women with previous GAS infection prior to invasive as well as semi-invasive gynecological or obstetric procedures.

Persistence of levator ani sonographic defect detected by three-dimensional transperineal sonography in primiparous women.

OBJECTIVES: Correlation of the sonographic appearance of levator ani muscle (LAM) injury soon after delivery with that at long-term follow-up has not been described fully. We aimed to compare results of three-dimensional (3D) transperineal sonographic (TPS) evaluation of the LAM from the period immediately postpartum with long-term follow-up, to determine whether sonographic findings persist over time. METHODS: Primiparous women (n = 210) who had been examined by 3D-TPS in a previous study to determine LAM trauma 24-72 hours after delivery were invited to participate in a follow-up examination 3-21 months postpartum. We included in this study only women who were not pregnant when approached and who had not given birth in the interim. LAM trauma was diagnosed with 3D-TPS when we observed discontinuity and distortion of the most anteromedial part of the pubovisceral muscle in the coronal C-plane or rendered image. Initial and follow-up 3D-TPS results were compared using Cohen's kappa test for inter-rater agreement. RESULTS: Among the 87 women included in this study we found strong correlation between earlier and later sonographic appearance of LAM: 17/21 women with a sonographic finding of LAM injury in the period immediately postpartum were positive in the follow-up examination, and only 2/66 women negative for LAM damage at the first examination were found to have sonographic evidence of LAM defect at follow-up (Cohen's kappa, 0.805 (95% CI, 0.656-0.954), P < 0.001). CONCLUSIONS: Our findings suggest that 3D-TPS of the LAM is a reliable examination. A sonographic finding of LAM defect identified in the period immediately postpartum persists months or years after delivery; therefore, this test may be performed following delivery, or may be delayed without impacting the result. It is likely that this sonographic defect represents real anatomical disruption and is not an imaging artifact.

Sonographic finding of postpartum levator ani muscle injury correlates with pelvic floor clinical examination.

OBJECTIVES: Correlation of the sonographic finding of levator ani muscle (LAM) injuries with clinical examination in primiparous women following vaginal delivery has not been fully described. We aimed to examine the correlation of three-dimensional transperineal ultrasound (3D-TPS) finding of LAM defects with results of clinical examination of the pelvic floor, at intermediate follow-up. METHODS: Subjects were primiparae 3-21 months following vaginal delivery, who had not become pregnant or delivered in the interim. On 3D-TPS, LAM trauma was diagnosed when discontinuity and distortion were visible in the most anteromedial part of the pubovisceral muscle in the coronal C-plane or rendered image. Clinical examination was performed by a physiotherapist who was blinded to the ultrasound results, and included palpation of the medial and lateral parts of the LAM mass, evaluation of tissue quality and whether there was any palpable gap. Muscle strength was evaluated using the modified Oxford scale. RESULTS: Eighty-seven women were included, 19 (21.8%) of whom were found to have a sonographic LAM injury. Oxford score palpation parameter of asymmetric muscle mass or texture was significantly correlated with the finding of a LAM defect: of 68 women with normal 3D-TPS, 22 (32.4%) were found to have asymmetry of muscle mass or tissue quality on clinical examination vs 12 (63.2%) of 19 women with sonographic evidence of LAM injury (P = 0.016). Muscle strength and endurance parameters did not significantly correlate with the 3D-TPS findings. CONCLUSION: Our findings suggest that persistent 3D-TPS LAM injury after primary vaginal delivery has clinical expression in changes in mass and texture of the LAM, as assessed by palpation.

[Interventional endovascular therapy of infrarenal abdominal aortic aneurysm]. / Die interventionelle/endovaskuläre Therapie des infrarenalen abdominellen Aortenaneurysmas.

The catheter-based interventional therapy (endovascular aortic repair EVAR) of abdominal aortic aneurysms (AAA) has gained an established place in the spectrum of therapeutic options. The procedure is characterized by low peri-interventional morbidity and mortality. Multislice computed tomography (CT) has a dominant role in defining the correct indications and in selecting an appropriate stent graft prior to the intervention. The rate of acute conversions could be reduced from 2.9 % to 0 % in our own elective patient population since 2010. In our vascular centre the proportion of patients treated by EVAR was 39.5 % (102 out of 258). The procedure is used routinely in patients who have an increased risk for general anesthesia or open surgery due to concomitant diseases. It is also used in patients with a reduced local operability due to prior surgery, abdominal diseases or radiation therapy. Arterial closure devices allow a completely percutaneous approach in a certain group of patients. However, after EVAR a life-long surveillance is mandatory because delayed therapy failure has been described. In younger patients who do not have a higher risk open surgery is still an option. The paper describes techniques, results und complications of EVAR.

Tissue microarray-based study of patients with lymph node-positive breast cancer shows tyrosine phosphorylation of signal transducer and activator of transcription 3 (tyrosine705-STAT3) is a marker of good prognosis

BACKGROUND: Although lymph node-positive breast cancers are associated with poorer prognosis, individual patients may have different clinical outcomes. Signal transducer and activator of transcription 3 (STAT3) is a point of convergence for numerous oncogenic signalling pathways. The goal of this study was to determine the prognostic value of phosphorylated (tyrosine705)-STAT3 in node-positive breast cancer patients. METHODS: Immunohistochemical analysis of Phospho- STAT3 was performed on a tissue microarray of breast cancer specimens. The expression pattern of Phospho-STAT3 was correlated with survival outcome, and clinical and pathological parameters. RESULTS: Out of 125 interpretable tumours, positive Phospho- STAT3 nuclear expression was seen in 35 (28%) of tumours. There was no significant relationship between Phospho-STAT3 expression and clinical-pathological parameters including age, hormonal receptor status, grade and tumour size. Interestingly positive tumours had a significantly improved disease-free survival at 5 years (p=0.035). Additionally, positive Phospho-STAT3 nuclear expression was correlated with significantly improved survival at both 5 years (p=0.023) and 10 years (p=0.026). Finally, in multivariate analyses Phospho-STAT3 was found to be an independent prognostic marker of overall survival in node-positive breast cancer patients. CONCLUSION: These findings support the role of Phospho- STAT3 as an important independent prognostic marker in node-positive breast cancer patients (AU)

[Chronic intestinal ischemia]. / Chronische intestinale Ischämie.

Ischemic lesions of the splanchnic organs are characterized by an insidious course and therefore are often underestimated. They can result in dramatic courses of disease which even in the last decade still results in a mortality of up to 90%. The reasons for this depressing situation are various but mainly due to insufficient consideration of the symptoms and late therapy due to delayed diagnosis.The incidence of chronic splanchnic ischemia is approximately 1-2% of all abdominal diseases. In contrast to acute intestinal ischemia the course is progressive, caused by progression of the underlying atherosclerosis and polymorbidity in this aging society. On the one hand occlusions of splanchnic arteries are diagnosed more often and on the other hand the incidence has increased due to the rising number of therapy-linked vascular catheter maneuvers. Due to excellent collateralization, diffuse stenotic processes can maintain asymptomatic for a long time. Duplex sonography should be performed as this technique reveals relevant insights into the hemodynamic severity of lesions.

[Adjusting a surgery department to new conditions in the German health service: day surgery, short-stay surgery, vascular centers and wound centers]. / Anpassung einer chirurgischen Klinik an die Herausforderungen im GesundheitswesenDie Tageschirurgie, die Kurzzeitchirurgie, das Gefässzentrum und die Wundsprechstunde.

In April 1994, after the preconditions for ambulatory surgery came into effect, the day surgery ward was opened in the Hamburg-Harburg General Hospital. Besides ambulatory operations, this ward provides all surgical pre- and postoperative care. In addition to the surgical sections, different departments (urology, gynecology, dentistry, radiology, ophthalmology) take advantage of the ward. As preoperative diagnostics also could be completed ambulatorily, we were then able to establish a short-stay surgery department. This ward was opened in June 1996. One year later, the vascular center ("Gefässcentrum Harburg") was established by the departments for surgery, radiology, and angiology. This structure has been integrated into the day and short-stay surgery wards functionally and spatially. With the organizational structures mentioned and by setting up a wound consultation service, it is possible to optimize the capacity for admissions, operations, and beds. Government and health insurance demands for better processing can be filled and efficiency is increased.

The mutant distribution of an RNA species replicated by Q beta replicase.

The mutant spectrum of an RNA species that is replicated by Q beta replicase, MNV-11, was investigated by retrotranscribing the RNA to DNA and cloning it into plasmids. The sequences of several cDNA clones of MNV-11 populations amplified by Q beta replicase under various conditions were determined and compared. A surprisingly broad mutant distribution was found: the consensus sequence never made up more than 40% of the total population and was accompanied by many mutants. Most mutants had several base exchanges, insertions and/or deletions; up to nine of the total 86 nucleotides were changed. The mutants found had replication rates comparable to that of the wild-type and were thus enriched in the population by selection forces. When the growth conditions were changed, the mutant distribution centre was shifted. The published consensus sequence of MNV-11 did not have the highest growth rate of the mutants, but was rather the best adapted to the various selection forces governing the growth phases the replicating RNA went through, i.e. it had found an optimal compromise between the rates of overall replication, enzyme binding and double strand formation.

[Critical ischemia of the extremities caused by ergotism. Treatment with intra-arterial prostaglandin E1 infusion]. / Kritische Extremitätenischämie durch Ergotismus. Behandlung mit intraarterieller Prostaglandin-E1-Infusion.

Severe ischaemia of the left upper limb developed in a 42-year-old woman (who had suffered from migraine since the age of 6 years) after the intake of 12 mg ergotamine tartrate (six suppositories of Cafergot within 5 hours). The left hand became very painful and pale with loss of touch sensation, and she could not move her fingers. Selective catheter angiography demonstrated typical signs of vascular ergotism: arterial spasms, corkscrew-like collaterals and segmental arterial occlusions. The patient's symptoms began to improve 10 minutes after starting an intraarterial infusion of prostaglandin E1 (0.34 ng/kg per min over 10 hours). Sensory function in the fingers was restored after 24 hours and reactive hyperaemia had occurred. Radiological examination after 48 hours showed complete recovery. This case emphasizes the need for obtaining an exact history regarding drug intake in any case of acute peripheral vascular disorder, but especially if there is no pointer to arterial thromboembolism. Angiography is of value in the differential diagnosis of suspected ergotism. Intraarterial infusion of prostaglandin E1 has few side effects and is immediately effective.

Cloning, restriction digestion and DNA labeling of large DNA fragments (greater than or equal to 1 kb) in the presence of remelted SeaPlaque GTG agarose gels.

Large DNA fragments (greater than or equal to 1 kb), separated in low melting temperature SeaPlaque GTG agarose gels, can be enzymatically processed directly in the presence of this agarose (in-gel). Time saving protocols are discussed for in-gel processing of large DNA fragments in the presence of remelted SeaPlaque GTG agarose, including cloning into pUC18, nick translation, random priming and restriction digestion. These in-gel molecular biology techniques are as efficient as those using DNA recovered from agarose. The effects of UV irradiation, Mg2+ concentration and agarose concentration on selected in-gel protocols are also discussed.

Nucleotide sequence analysis of the purEK operon encoding 5'-phosphoribosyl-5-aminoimidazole carboxylase of Escherichia coli K-12.

5'-Phosphoribosyl-5-aminoimidazole (AIR) carboxylase (EC 4.1.1.21) catalyzes step 6, the carboxylation of AIR to 5'-phosphoribosyl-5-aminoimidazole-4-carboxylic acid, in the de novo biosynthesis of purine nucleotides. As deduced from the DNA sequence of restriction fragments encoding AIR carboxylase and supported by maxicell analyses, AIR carboxylase was found to be composed of two nonidentical subunits. In agreement with established complementation data, the catalytic subunit (deduced Mr, 17,782) was encoded by the purE gene, while the CO2-binding subunit (deduced Mr, 39,385) was encoded by the purK gene. These two genes formed an operon in which the termination codon of the purE gene overlapped the initiation codon of the purK gene. The 5' end of the purEK mRNA was determined by mung bean nuclease mapping and was located 41 nucleotides upstream of the proposed initiation codon. The purEK operon is regulated by the purR gene product, and a purR regulatory-protein-binding site related to the sequences found in other pur loci was identified in the purEK operon control region.

A rat histone H4 gene closely associated with the testis-specific H1t gene.

A rat histone H4 gene closely associated with the testis-specific H1t gene was isolated by screening the Sargent-Bonner rat genomic library using cloned human histone genes as probes. Both the H4 gene and the H1t gene are located on a 7-kb EcoRI genomic DNA fragment. Although the deduced amino acid sequence of the rat H4 histone is identical to that of the sequence of human histone H4, the nucleotide sequence of the coding region differs significantly from the coding region of the human H4 gene. Moreover, the relative spacing between the 5'-consensus sequence elements is unique for an H4 gene. S1-nuclease protection analyses reveal that both the H4 and H1t mRNA species are present in a fraction of rat testis cells highly enriched in pachytene spermatocytes, while only the H4 mRNA species is present in a rat myeloma cell line (Y3-Ag1.2.3). During a 1-h hydroxyurea treatment of the Y3 cells, which produces a 99% inhibition of DNA synthesis, the level of this H4 mRNA drops by only 50%, indicating that the stability of this mRNA is only partially coupled with DNA synthesis.

Identification and nucleotide sequence of a gene encoding 5'-phosphoribosylglycinamide transformylase in Escherichia coli K12.

5'-Phosphoribosylglycinamide transformylase (EC 2.1.2.2), encoded by the purN gene of Escherichia coli, catalyzes the synthesis of 5'-phosphoribosylformylglycinamide from 5'-phosphoribosylglycinamide (GAR). The mature protein, as deduced from the purN structural gene sequence, contains 212 amino acid residues and has a calculated Mr of 23,241. The purN gene is located adjacent to and immediately downstream from the purM gene encoding 5'-phosphoribosyl-5-aminoimidazole (AIR) synthetase where the initiation codon for GAR transformylase overlaps the termination codon of AIR synthetase. Based on polarity studies, the expression of the purN gene originates from the purM control region and thus forms a purMN operon. The E. coli GAR transformylase shows greater homology to the GAR transformylase domain of the trifunctional Gart polypeptide of Drosophila than to the single GAR transformylase of Saccharomyces. Immediately downstream from the purN gene of the purMN operon is a region of dyad symmetry capable of forming a hairpin stem and loop structure characteristic of a rho-independent terminator.