Trio exome sequencing is highly relevant in prenatal diagnostics.

OBJECTIVE: About 3% of newborns show malformations, with about 20% of the affected having genetic causes. Clarification of genetic diseases in postnatal diagnostics was significantly improved with high-throughput sequencing, in particular through whole exome sequencing covering all protein-coding regions. Here, we aim to extend the use of this technology to prenatal diagnostics. METHOD: Between 07/2018 and 10/2020, 500 pregnancies with fetal ultrasound abnormalities were analyzed after genetic counseling as part of prenatal diagnostics using WES of the fetus and parents. RESULTS: Molecular genetic findings could explain ultrasound abnormalities in 38% of affected fetuses. In 47% of these, disease-causing de novo variants were found. Pathogenic variants in genes with autosomal recessive or X-linked inheritance were detected in more than one-third (70/189 = 37%). The latter are associated with increased probability of recurrence, making their detection important for further pregnancies. Average time from sample receipt to report was 12 days in the recent cases. CONCLUSION: Trio exome sequencing is a useful addition to prenatal diagnostics due to its high diagnostic yield and short processing time (comparable to chromosome analysis). It covers a wide spectrum of genetic changes. Comprehensive interdisciplinary counseling before and after diagnostics is indispensable.

Kagami-Ogata syndrome: an important differential diagnosis to Beckwith-Wiedemann syndrome.

We report the case of a fetus with sonographic characteristics of Beckwith-Wiedemann syndrome (BWS). A 30-year-old gravida 2 para 1 was referred to our fetal medicine unit with an omphalocele. Fetal macrosomia, organomegaly, and polyhydramnios but no macroglossia were detected and BWS was suspected. Genetic testing for BWS did not confirm the suspected diagnosis as the karyotype was normal. Symptomatic polyhydramnios led to repeated amnioreductions. At 35 + 5 weeks of gestation, a female neonate of 3660 g was delivered with APGAR scores of 6/7/8, after 1/5/10 min, respectively. The abnormal shape of the thorax, facial dysmorphism, need for ventilation, and generalized muscular hypotonia led to the suspicion of Kagami-Ogata syndrome (KOS), which was confirmed by genetic testing. KOS in our patient was caused by a large deletion in the MEG3-region on chromosome 14q32 affecting the maternal allele. In this report, we highlight the notion that when sonographic signs suggestive of BWS such as macrosomia, polyhydramnios, and omphalocele are present and genetic testing does not confirm the suspected diagnosis, KOS should be tested for.

A de novo nonsense mutation in ZBTB18 plus a de novo 15q13.3 microdeletion in a 6-year-old female.

ZBTB18 has been proposed as candidate gene for microcephaly and abnormalities of the corpus callosum based on overlapping microdeletions of 1q43q44. More recently, de novo mutations of ZBTB18 have been identified in patients with syndromic and non-syndromic intellectual disability. Heterozygous microdeletions of 15q13.3 encompassing the candidate gene CHRNA7 are associated with developmental delay or intellectual disability with speech problems, hypotonia, and seizures. They are characterized by significant variability and reduced penetrance. We report on a patient with a de novo ZBTB18 nonsense mutation and a de novo 15q13.3 microdeletion, both in a heterozygous state, identified by next generation sequencing and array-CGH. The 6-year-old girl showed global developmental delay, absent speech, therapy-refractory seizures, ataxia, muscular hypotonia, and discrete facial dysmorphisms. Almost all of these features have been reported for both genetic aberrations, but the severity could hardly been explained by the microdeletion 15q13.3 alone. We assume an additive effect of haploinsufficiency of ZBTB18 and CHRNA7 in our patient. Assembling the features of our patient and the published patients, we noted that only one of them showed mild anomalies of the corpus callosum. Moreover, we hypothesize that nonsense mutations of ZBTB18 are associated with a more severe phenotype than missense mutations. This report indicates that haploinsufficiency of additional genes beside ZBTB18 causes the high frequency of corpus callosum anomalies in patients with microdeletions of 1q43q44 and underlines the importance of an NGS-based molecular diagnostic in complex phenotypes.

A novel mutation in NEB causing foetal nemaline myopathy with arthrogryposis during early gestation.

Nemaline myopathies are a clinically and genetically heterogeneous group of congenital myopathies, mainly characterized by muscle weakness, hypotonia and respiratory insufficiency. Here, we report a male foetus of consanguineous parents with a severe congenital syndrome characterized by arthrogryposis detected at 13 weeks of gestation. We describe severe complex dysmorphic facial and musculoskeletal features by post mortem fetal examination confirming the prenatal diagnosis. Histomorphological and ultrastructural studies of skeletal muscle reveal mini-rods in myotubes caused by a novel homozygous splice-site mutation in NEB (NM_001164508, chr2:g.152,417,623C>A GRCh37.p11 | c.19,102-1G>T ENST00000397345.3). No rods were seen in the myocardium. We discuss the relevance of this mutation in the context of nemaline myopathies associated with early developmental musculoskeletal disorders.

Synchronous adenocarcinomas of intestinal type of the inner nose and the colon.

Intestinal types of adenocarcinoma of the inner nose and colorectal adenocarcinoma present a stupendous similarity of morphological and immunohistochemical features. The previously unpublished observation of a synchronous manifestation of both adenocarcinoma enabled us to compare the tumors using molecular and immunohistochemical methods. Polymerase chain reaction was performed in order to investigate microsatellite instability. Mutation of p53 and K-ras was examined by direct DNA sequencing. Chromosomal imbalances were investigated by comparative genomic hybridization. Histology and immunohistochemical reactions were nearly identical. PCR results revealed no microsatellite instability or loss of heterozygosity in any of the tumors. A p53 mutation in exon 5 could be detected in the colon tumor but not in the sinonasal carcinoma, while a K-ras mutation was only present in the tumor of the inner nose. The comparative genomic hybridization method revealed different chromosomal imbalances in the different tumors. Thus, the molecular pathologic data proved the presence of 2 independent primary adenocarcinomas of the intestinal type.

Hypoxic Preconditioning Increases Survival and Pro-Angiogenic Capacity of Human Cord Blood Mesenchymal Stromal Cells In Vitro.

Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) upon transplantation in ischemic tissue. Given the interest in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro-angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity) and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC) were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, "post-ischemic" cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic preconditioning might be a translationally relevant strategy to increase the tolerance of cord blood MSCs to ischemia and improve their therapeutic efficacy in clinical applications.

PHOTOPROBE biotin: an alternative method for labeling archival DNA for comparative genomic hybridization.

Comparative genomic hybridization (CGH) represents a powerful method for screening the entire genome of solid tumors for chromosomal imbalances. Particularly it enabled the molecular cytogenetic analysis of archival, formalin-fixed, paraffin-embedded (FFPE) tissue. A well-known dilemma, however, is the poor DNA quality of this material with fragment sizes below 1000 bp. Nick translation, the conventionally used enzymatic DNA labeling method in CGH, leads to even shorter fragments often below a critical limit for successful analysis. In this study we report the alternative application of non-enzymatic, PHOTOPROBE biotin labeling for conjugation of the hapten to the DNA prior to in situ hybridization and fluorescence detection. We analyzed 51 FFPE tumor samples mainly from the upper respiratory tract by both labeling methods. In 19 cases, both approaches were successful. The comparison of hybridized metaphases showed a distinct higher fluorescence signal of the PHOTOPROBE samples sometimes with a discrete cytoplasm background which however did not interfere with specificity and sensitivity of the detected chromosomal imbalances. For further 32 cases characterized by an average DNA fragment size below 1000 bp, PHOTOPROBE biotin was the only successful labeling technique thus offering a new option for CGH analysis of highly degraded DNA from archival material.

Bub1 regulates chromosome segregation in a kinetochore-independent manner.

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458-476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis.

Hydrometrocolpos, postaxial polydactyly, and hypothalamic hamartoma in a patient with confirmed Pallister-Hall syndrome: a clinical overlap with McKusick-Kaufman syndrome.

We present a preterm-born girl with polydactyly of both hands and massive hydrometrocolpos, the latter due to vaginal atresia. This association led initially to the diagnosis of McKusick-Kaufman syndrome (MKKS). However, additional features, including characteristic radiographic findings of the hands and a large hypothalamic tumour, presumably a hamartoma, favoured the diagnosis of Pallister-Hall syndrome (PHS), which was then genetically confirmed by detection of a GLI3 mutation (Q717X). This is the second genetically confirmed case revealing the previously described association of PHS with hydrometrocolpos due to vaginal atresia as a clinical overlap with MKKS.

Chromosomal imbalances in wood dust-related adenocarcinomas of the inner nose and their associations with pathological parameters.

Comparative genomic hybridization (CGH) was used to screen 42 wood dust-related sinonasal adenocarcinomas for chromosomal alterations. The tumour collection comprised 39 papillary-tubular cylinder cell adenocarcinomas (PTCCs; six cases G1, 23 G2, and ten G3), two alveolar goblet cell adenocarcinomas (AGCs), and one signet ring cell adenocarcinoma (SRC), according to the Kleinsasser and Schroeder classification. Copy number changes were detected in 41 tumours (97.6%). The one carcinoma without imbalances was a PTCC-G1. DNA gains were most frequently seen on chromosomes 12p (83%), 7q (74%), 8q (71%), and 20q (71%), 11q (61%), 22 (59%), and 1q (52%). Pronounced overrepresentations suggestive of high copy amplifications were detected on 8q (15 cases, 36%), 7q (six cases, 14%), 20q (five cases, 12%), 13q14 (three cases, 7%), 1q22, 5p, 12p and 20 (two cases, 5% each), and 2q24, 3q13, 3q22, 7p, 14q12, and 16q13 (one case, each 2%). Frequent chromosomal losses occurred at 5q (81%), 18q (76%), 4 (74%), 8p (61%), 9p (60%), 6q and 17p (52% each), and 3p, 13q, and 21 (50% each). There was a quantitative as well as a qualitative increase of alterations from PTCC-G1 to PTCC-G2 and finally PTCC-G3, confirming the usefulness of histopathological grading. While PTCC-G1 carried only a few alterations, namely gains on chromosomes 17 and 7 as well as losses of 4q and 13q, PTCC-G2 already carried many of the above-mentioned alterations, while PTCC-G3 showed significantly more gains of 7q, 8q, and 12p, and losses of 8p and 17p. Additionally, the latter subgroup was particularly prone to carry pronounced DNA gains. These data provide further evidence for a recurrent pattern of chromosomal imbalances in sinonasal adenocarcinomas and highlight distinct aberrations that are associated with tumour differentiation and progression.