Genetic and functional analyses detect one pathological NFATC1 mutation in a Chinese tricuspid atresia family.

BACKGROUND: Cardiac valvulogenesis is a highly conserved process among vertebrates and cause unidirectional flow of blood in the heart. It was precisely regulated by signal pathways such as VEGF, NOTCH, and WNT and transcriptional factors such as TWIST1, TBX20, NFATC1, and SOX9. Tricuspid atresia refers to morphological deficiency of the valve and confined right atrioventricular traffic due to tricuspid maldevelopment, and is one of the most common types of congenital valve defects. METHODS: We recruited a healthy couple with two fetuses aborted due to tricuspid atresia and identified related gene mutations using whole-exome sequencing. We then discussed the pathogenic significance of this mutation by bioinformatic and functional analyses. RESULTS: PROVEAN, PolyPhen, MutationTaster, and HOPE indicated the mutation could change the protein function and cause disease; Western blotting showed the expression of NFATC1 c.964G>A mutation was lower than the wild type. What's more, dual-luciferase reporter assay showed the transcriptional activity of NFATC1 was impact by mutation and the expression of downstream DEGS1 was influenced. CONCLUSION: Taken together, the c.964G>A mutation might be pathological and related to the occurrence of disease. Our research tended to deepen the understanding of etiology of tricuspid atresia and gene function of NFATC1, and provide some references or suggestions for genetic diagnosis of tricuspid atresia.

Prenatal case of Simpson-Golabi-Behmel syndrome with a de novo 370Kb-sized microdeletion of Xq26.2 compassing partial GPC3 gene and review.

BACKGROUND: Simpson-Golabi-Behmel syndrome type 1 (SGBS1) is a rare X-linked recessive disorder characterized by pre- and postnatal overgrowth and a broad spectrum of anomalies including craniofacial dysmorphism, heart defects, renal, and genital anomalies. Due to the ultrasound findings are not pathognomonic for this syndrome, most clinical diagnosis of SGBS1 are made postnatally. METHODS: A pregnant woman with abnormal prenatal sonographic findings was advised to perform molecular diagnosis. Single nucleotide polymorphism array (SNP array) was performed in the fetus, and the result was validated with multiplex ligation-dependent probe amplification (MLPA) and real-time quantitative PCR (qPCR). RESULTS: The prenatal sonographic presented with increased nuchal translucency at 13 gestational weeks, and later at 21 weeks with cleft lip and palate, heart defect, increased amniotic fluid index and over growth. A de novo 370Kb-deletion covering the 5'-UTR and exon 1 of GPC3 gene was detected in the fetus by SNP array, which was subsequently confirmed by MLPA and qPCR. CONCLUSION: The de novo 370Kb hemizygous deletion of 5'-UTR and exon 1 of GPC3 results in the SGBS1 of this Chinese family. Combination of ultrasound and genetics tests helped us effectively to diagnose the prenatal cases of SGBS1. Our findings also enlarge the spectrum of mutations in GPC3 gene.

The VANGL1 P384R variant cause both neural tube defect and Klippel-Feil syndrome.

BACKGROUND: Neural tube defect (NTD) is a common birth defect causing much death in the world. Variants in VANGL1 lead to NTD and caudal regression syndrome. NTD displays a complex phenotype encompassing both genetic and environmental factors. METHODS: The fetus was diagnosed by prenatal ultrasound examination. Postnatal CT and autopsy were performed. Genetic testing was conducted in the family and Sanger sequencing was validated. Multiple prediction soft-wares were used to predict the pathogenicity of the variant. RESULTS: The VANGL1 gene variant c.1151C>G (P384R) was detected in a fetus diagnosed with tethered spinal cord and sacrococcygeal lipoma. The VANGL1 variant c.1151C>G (P384R) was reported in a Klippel-Feil syndrome patient. The VANGL1 variant was validated in the trio-family but the mother showed no abnormalities. CONCLUSION: Overall, this study presents fetal NTD caused by the same VANGL1 variant found in a Klippel-Feil syndrome patient with complete clinical information of prenatal ultrasound, postnatal CT, and genetic results as early as 25 GW. Our study not only expands the VANGL1 mutational spectrum but also sheds light on the important role of the VANGL1 P384R variant in human development.

CEP135 associated primary microcephaly-A rare presentation in early second trimester.

Primary microcephaly (MCPH) is a rare neurogenic disorder with most cases being inherited in an autosomal recessive pattern. The present report is of a case of second gravid patient with recurrent fetal microcephaly with agenesis of corpus callosum, cerebellar hypoplasia and ventriculomegaly. Maternal TORCH profile and amniotic fluid chromosomal microarray were normal. Following the termination of pregnancy, the autopsy evaluation has shown additional findings of evolving craniosynostosis, and semilobar holoprosencephaly. Whole exome sequencing done on fetal DNA from amniotic fluid, revealed a pathogenic compound heterozygous variant (NM_025009.5) c.2863C>T (p.Arg955Ter) in exon 22 and c.1372_1375del (p.Lys459SerfsTer2) in exon 11 of CEP135 gene: known to cause primary microcephaly-8; and both partners in the couple are heterozygous carriers for the same. With the identification of MCPH genes and with the availability of next-generation sequencing (NGS) based exome sequencing, a definitive prenatal diagnosis of primary microcephaly and also appropriate genetic counselling for the couple has become possible.

Non-classic splicing mutation in the CPLANE1 (C5orf42) gene cause Joubert syndrome in a fetus with severe craniocerebral dysplasia.

BACKGROUD: Joubert syndrome is a rare neurodevelopmental disorder characterized by clinical and genetic heterogeneity. The characteristic molar tooth sign, which resulted from cerebellar vermis hypoplasia and midbrain anomalies, is expected to be the key diagnostic feature for this disease. However, it is not easy to make a definite diagnosis in prenatal only based on the imageology due to its clinical heterogeneity. CASE REPORT: We report on a fetus who was detected cerebellum dysplasia and encephalocele by ultrasound at 19 and 23 gestational weeks and confirmed by MRI examination. The pregnancy was terminated at 23 weeks of gestation. Postaxial polydactyly and deficiency in occipital bone and skin were identified in the induced fetus. RESULTS: The whole exome sequencing identified a novel compound heterozygous variation in the CPLANE1 gene related with Joubert syndrome, including a 2-bp insertion, NM_023073.3:c.1383_1384dup; p.(Gly462Glufs*3) and a non-classic splicing variation, NC_000005.10(NM_023073.3):c.7691-5_7691-4del. The pathogenicity of the non-classic splicing variation was further confirmed by cDNA level sequencing, which showed a exon 39 skipping that would introduce a premature termination. The novel compound heterozygous variation caused a complete function loss of the CPLANE1 gene. CONCLUSION: The cerebellum dysplasia fetus without obvious molar tooth sign was finally diagnosed as Joubert syndrome, combined with genetic detecting and the postnatal clinical symptoms. We also highlight the clinical heterogeneity of encephalodysplasia in Joubert syndrome, which increases the clinical diagnosis difficulty, especially for prenatal diagnosis. Our findings provided a new perspective for the prenatal diagnosis of Joubert syndrome with severe craniocerebral dysplasia and expanded the variation spectrum of the CPLANE1 gene.

Loss-of-function or gain-of-function variations in VINCULIN (VCL) are risk factors of human neural tube defects.

BACKGROUND: Neural tube defects (NTDs) are severe birth defects resulting from the failure of neural tube closure during embryogenesis. Both genetic and environmental factors contribute to the occurrence of NTDs and the heritability of NTDs is approximately 70%. As a key component of focal adhesions, Vinculin (VCL) plays pivotal roles in cell skeleton remodeling and signal transduction. Vcl deficient mice displayed NTD, but how VCL variants contribute to human NTDs has not been addressed yet. METHODS: We screened VCL variants in a Chinese cohort of 387 NTDs and 244 controls by targeted next-generation sequencing. RESULTS: We identified four case-specific VCL variations (p.M209L, p.D256fs, p.L555V and p.R586Q). VCL p.D256fs and p.L555V are novel variations that have never been reported. Our analysis revealed that p.D256fs is a loss-of-function variant, while p.L555V showed a gain of function in planner cell polarity (PCP) pathway regulation and cell migration, probably due to its enhanced protein stability. CONCLUSION: Our study reports human NTD specific novel variations in VCL and provides the functional evaluation of VCL variants related to the etiology of human NTDs.

TP63-mutation as a cause of prenatal lethal multicystic dysplastic kidneys.

BACKGROUND: Ectrodactyly-ectodermal dysplasia-clefting syndrome 3 (EEC) is one of the six overlapping syndromes caused by mutations in the tumor protein p63 gene (TP63). EEC is suspected when patients have cleft hands or feet, polydactyly, and syndactyly, abnormal development of the ectodermally derived structures, and orofacial clefting. Genitourinary (GU) anomalies have been identified in patients with EEC, yet these are often under-recognized and under-reported. The available literature on sonographic prenatal findings is sparse, especially when considering GU anomalies. METHODS: We present the case of a male stillborn fetus, who was found antenatally to have multicystic dysplastic kidneys and anhydramnios. Following the termination of pregnancy, examination and autopsy further revealed unilateral polydactyly and bilateral syndactyly which had not been previously identified on antenatal ultrasound. RESULTS: Whole-exome sequencing (WES) revealed a de novo heterozygous pathogenic variant in exon 5 of the TP63 gene: p.His247Arg: c.740A>G (NM_003722.4) which has been reported in the literature. The His247Arg variant has been published as a pathogenic variant in association with EEC, both with and without orofacial clefting. CONCLUSION: Our prenatal case expands the phenotypic spectrum of TP63-related disorders in general. In addition, it adds to the phenotype associated with the His247Arg pathogenic variant responsible for EEC. Further, we highlight the importance of WES as a postnatal tool to help clarify unexpected findings, and as a way to add to the spectrum of existing phenotypes of known single-gene disorders.

Monochorionic diamniotic twins of discordant external genitalia with 45,X/46,XY mosaicism.

BACKGROUND: Monozygotic twins with 45,X/46,XY mosaicism, discordant for phenotypic sex, are extremely rare. METHODS: This report describes the clinical findings of a rare case of 45,X/46,XY mosaicism in monozygotic twins with different external genitalia. Single nucleotide polymorphism (SNP) array analysis, performed by collecting DNA from each umbilical cord, showed identical SNPs in the autosomal chromosomes of both fetuses. RESULTS: Chorionic villus sampling of a 37-year-old primigravida carrying monozygotic twins revealed a 45,X/46,XY karyotype. Autopsy of the aborted fetuses revealed a penis and testes on one fetus and a vagina, uterus, and ovaries in the other fetus--which also had severe cystic hygroma. Cell counting using fluorescence in situ hybridization with XY probes (XY-FISH) showed 20% and 80% abundance of 45,X cells in the internal genitalia, liver, heart, lung, adrenal gland, bone marrow, and spine of the male and female fetuses, respectively. CONCLUSION: These results indicated that the fetuses were genetically monozygotic twins and their different degrees of mosaicism may have resulted in different genital phenotypes.

Bronchogenic cyst presenting as content of omphalocele - A case report.

Bronchogenic cyst (BC) is a very rare congenital anomaly occurring due to budding of the primitive foregut, and its common location is the posterior mediastinum. BC when diagnosed prenatally can be treated if it is encroaching on the development of lungs. BC has been reported in other locations such as cervical, thoracic, abdominal sites and also as subcutaneous lesions. Omphalocele is a congenital malformation occurring due to a central defect in the abdominal wall with herniation of the viscera. The nonentity documented here was found in a female fetus with 20 weeks of gestational age. The mother was a primigravida who had antenatal ultrasound scan rendering diagnosis of a live fetus having abdominal wall defect with omphalocele. This case is exceptionally rare as the content of omphalocele was BC having a classical wall lined by pseudostratified ciliated columnar epithelium overlying band-like cartilage. The extensive search in the literature did not reveal another similar case.

Evaluation of the stapedial tendon growth dynamic in human fetuses.

PURPOSE: The main objective of the study was to investigate the morphometric properties of the stapedial tendon (ST) for pediatric otosurgeons and anatomists. METHODS: The present study was placed on 15 fetuses (8 females, 7 males) aged from 20 to 30 weeks of gestation (at mean, 24.27 ± 3.24 weeks) using the collection of the Anatomy Department of Medicine Faculty, Mersin University. All measurements were obtained with a digital image analysis software. RESULTS: In terms of male/female or right/left comparisons, no statistically significant difference was found in relation with the numerical data of ST. The surface area, length, and width of ST were detected as follows: 0.61 ± 0.15 mm2, 1.27 ± 0.30 mm, and 0.45 ± 0.08 mm, respectively. The absence of ST was observed in two fetuses with and without severe malformations. In another fetus with cleft lip and polydactyly, multiple abnormalities were bilaterally identified in the middle ear: (1) the absence of the incudostapedial joint and (2) the presence of an abnormal tissue attaching to the stapes. The abnormal tissue was determined to be irregular dense connective tissue using light microscope and electron microscope. CONCLUSION: Our findings showed that ST did not proportionally grow according to increasing gestational weeks. In the light of the numerical data, we thought that similar to stapes, ST attains the adult size in the fetal period. As ST anomalies may accompany severe malformations (e.g., cleft lip, polydactyly or syndactyly) that can be easily detected on observation by clinicians, we suggest that the detailed examination of middle ear in newborns should be taken into account for early diagnosis of conductive hearing loss to prevent any management delays.

Characterization of three ciliopathy pedigrees expands the phenotype associated with biallelic C2CD3 variants.

Whole exome sequencing (WES) is utilized in diagnostic odyssey cases to identify the underlying genetic cause associated with complex phenotypes. Recent publications suggest that WES reveals the genetic cause in ~25% of these cases and is most successful when applied to children with neurological disease. The residual 75% of cases remain genetically elusive until more information becomes available in the literature or functional studies are pursued. WES performed on three families with presumed ciliopathy diagnoses, including orofaciodigital (OFD) syndrome, fetal encephalocele, or Joubert-related disorder, identified compound heterozygous variants in C2CD3. Biallelic variants in C2CD3 have previously been associated with ciliopathies, including OFD syndrome type 14 (OFD14; MIM: 615948). As three of the six identified variants were predicted to affect splicing, exon-skipping analysis using either RNA sequencing or PCR-based methods were completed to determine the pathogenicity of these variants, and showed that each of the splicing variants led to a frameshifted protein product. Using these studies in combination with the 2015 ACMG guidelines, each of the six identified variants were classified as either pathogenic or likely pathogenic, and are therefore likely responsible for our patients' phenotypes. Each of the families had a distinct clinical phenotype and severity of disease, extending from lethal to viable. These findings highlight that there is a broad phenotypic spectrum associated with C2CD3-mediated disease and not all patients present with the typical features of OFD14.

New splicing pathogenic variant in EBP causing extreme familial variability of Conradi-Hünermann-Happle Syndrome.

X-linked dominant chondrodysplasia punctata (CDPX2 or Conradi-Hünermann-Happle syndrome, MIM #302960) is caused by mutations in the EBP gene. Affected female patients present with Blaschkolinear ichthyosis, coarse hair or alopecia, short stature, and normal psychomotor development. The disease is usually lethal in boys. Nevertheless, few male patients have been reported; they carry a somatic mosaicism in EBP or present with Klinefelter syndrome. Here, we report CDPX2 patients belonging to a three-generation family, carrying the splice variant c.301 + 5 G > C in intron 2 of EBP. The grandfather carries the variant as mosaic state and presents with short stature and mild ichthyosis. The mother also presents with short stature and mild ichthyosis and the female fetus with severe limb and vertebrae abnormalities and no skin lesions, with random X inactivation in both. This further characterizes the phenotypical spectrum of CDPX2, as well as intrafamilial variability, and raises the question of differential EBP mRNA splicing between the different target tissues.

Biallelic loss of function variants in COASY cause prenatal onset pontocerebellar hypoplasia, microcephaly, and arthrogryposis.

Pontocerebellar hypoplasia (PCH) is a heterogeneous neurodegenerative disorder with a prenatal onset. Using whole-exome sequencing, we identified variants in the gene Coenzyme A (CoA) synthase (COASY) gene, an enzyme essential in CoA synthesis, in four individuals from two families with PCH, prenatal onset microcephaly, and arthrogryposis. In family 1, compound heterozygous variants were identified in COASY: c.[1549_1550delAG]; [1486-3 C>G]. In family 2, all three affected siblings were homozygous for the c.1486-3 C>G variant. In both families, the variants segregated with the phenotype. RNA analysis showed that the c.1486-3 C>G variant leads to skipping of exon 7 with partial retention of intron 7, disturbing the reading frame and resulting in a premature stop codon (p.(Ala496Ilefs*20)). No CoA synthase protein was detected in patient cells by immunoblot analysis and CoA synthase activity was virtually absent. Partial CoA synthase defects were previously described as a cause of COASY Protein-Associated Neurodegeneration (CoPAN), a type of Neurodegeneration and Brain Iron Accumulation (NBIA). Here we demonstrate that near complete loss of function variants in COASY are associated with lethal PCH and arthrogryposis.

An unnoticed variant of Sirenomelia with constellation of multiple anomalies

Sirenomelia is a rare fatal congenital anomaly, characterized by a single midline lower limb, urogenital anomalies, Potter's facies and a single umbilical artery. Approximately 400 cases have been reported in the literature. Based on the number of feet and the degree of lower limb bone fusion, it is classified into seven different types. Sirenomelia has been reported with associated anomalies involving multiple systems mainly of urogenital, respiratory as well as the alimentary tract system. In the present case, the authors reported an unnoticed variation in the fusion of lower limbs and its rare association with tracheoesophageal fistula

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Recomendaciones de buenas prácticas para el diagnóstico genético de abortos espontáneos e interrupciones voluntarias del embarazo por presentar defectos congénitos / Good practice recommendations in the molecular diagnosis of miscarriage and abortion due to multiple congenital malformations

Objetivos: La aparición de defectos congénitos produce una gran ansiedad en la familia y una enorme demanda asistencial. El objetivo principal radica en la redacción de las recomendaciones de buenas prácticas que sirvan de guía a los profesionales sanitarios para el diagnóstico clínico-genético de defectos congénitos. Metodología: El protocolo que proponemos contempla un modelo de actuación óptimo que incluye, la recogida de la información clínica inicial, la obtención de las muestras biológicas y los protocolos de actuación. Resultado: Se ha elaborado un modelo de historia clínica que ayude a la recogida de la información clínica pertinente. En la obtención de las muestras biológicas se aconseja la obtención de muestras fetales (de las 3 capas embrionarias) y muestras de los progenitores que serán procesarán teniendo en cuenta el algoritmo de actuación propuesto para el correcto diagnóstico genético del defecto congénito correspondiente. Conclusión: Esta guía recoge por primera vez, las recomendaciones de buenas prácticas para el diagnóstico genético de abortos con defectos congénitos

Aims: Congenital anomalies can cause anxiety within a family and high healthcare demand. The aim of this study was to write good practice recommendations to guide health professionals in the clinical-genetic diagnosis of congenital defects. Methods: The proposed protocol focuses on an optimal case scenario that includes collection of initial clinical data, biological sampling, and diagnostic algorithms. Results: A model of the optimal clinical history form was created to facilitate the collection of initial clinical data. For sampling, it is recommended to obtain at least one fetal sample (of the three embryonic germ layers). Moreover, samples from both parents should be taken to exclude mosaicism, following the diagnostic algorithm proposed for the correct genetic diagnosis of the corresponding congenital defect. Conclusion: This document is the first to gather good practice recommendations for the pre- and post-natal genetic diagnosis of miscarriages and abortions due to congenital defects

First fetal case of the 8q24.3 contiguous genes syndrome.

Molecular cytogenetics, particularly array-CGH, opened the way to the « genotype first approach ¼ and for the discovery of new micro rearrangement syndromes. This was the case for the 8q24.3 microdeletion syndrome. Here, we describe the phenotype of a fetus with a 8q24.3 deletion. This rare condition has to be considered as a contiguous genes syndrome because its phenotype is generated by the SCRIB and PUF60 adjacent gene endophenotypes. The fetus presented atrioventricular septal defect and hypoplastic aortic arch, facial dysmorphism, microretrognathia, dysmorphic ears, clinodactyly of the 5th digit on both hands, mild rocker bottom feet and abnormal third sacral vertebra. This fetus is the first case where the endophenotype produced by SCRIB gene is absent. This case is compared with the previous published cases.

Etiology of spontaneous abortion before and after the demonstration of embryonic cardiac activity in women with recurrent spontaneous abortion.

OBJECTIVE: To analyze the etiologic factors of spontaneous abortion in the first trimester among women with recurrent spontaneous abortion, specifically before and after the demonstration of embryonic cardiac activity. METHODS: A retrospective analysis included women with recurrent spontaneous abortion admitted to a center in Guangzhou, China, for dilation and curettage after a spontaneous abortion in the first trimester between January 2008 and December 2012. The etiologic factors of spontaneous abortion occurring before versus after the demonstration of cardiac activity were compared. RESULTS: A total of 232 women were included. Among 146 women with demonstrated cardiac activity before spontaneous abortion, 78 (53.4%) had an embryonic karyotype abnormality, 55 (37.7%) had traditional etiologic factors, and 34 (23.3%) had an unidentified cause. Among 86 women without cardiac activity, 41 (47.7%) had an embryonic karyotype abnormality, 28 (32.6%) had traditional etiologic factors, and 26 (30.2%) had an unidentified cause. After exclusion of abortions involving embryonic karyotype abnormalities, there was a higher incidence of APA positivity in the group with embryonic cardiac activity than in the other group (13/68 [19.1%] vs 1/45 [2.2%]; P=0.008) and a lower incidence of subclinical hypothyroidism (8/68 [11.8%] vs 12/45 [26.7%]; P=0.042). CONCLUSION: The distribution of etiologic factors in spontaneous abortion differs according to whether embryonic cardiac activity is recorded.