Chromosomal abnormalities associated with fetal pleural effusion (I): General overview.

Fetal pleural effusion has been reported to be associated with chromosomal abnormalities, genetic syndromes, obstructive uropathy, lymphatic vessel abnormalities such as Noonan syndrome, RASopathy and congenital lymphatic anomalies, thoracic cavity defects, Rh or ABO incompatibility, non-immune hydrops fetalis, infections, congenital cardiac anomalies, metabolic diseases and hematologic diseases such as α-thalassemia. This review provides an overview of chromosomal abnormalities associated with fetal pleural effusion which is useful for genetic counseling and fetal therapy at prenatal diagnosis of fetal pleural effusion.

Syndromic and single gene disorders associated with fetal pleural effusion (I): Noonan syndrome, RASopathy and congenital lymphatic anomalies.

Fetal pleural effusion has been reported to be associated with chromosomal abnormalities, genetic syndromes, obstructive uropathy, lymphatic vessel abnormalities such as Noonan syndrome, RASopathy and congenital lymphatic anomalies, thoracic cavity defects, Rh or ABO incompatibility, non-immune hydrops fetalis, infections, congenital cardiac anomalies, metabolic diseases and hematologic diseases such as α-thalassemia. This review provides an overview of syndromic and single gene disorders associated with fetal pleural effusion that is useful for genetic counseling and fetal therapy at prenatal diagnosis of fetal pleural effusion.

Chromosomal abnormalities associated with fetal pleural effusion (II): Specific and non-specific chromosome aberrations.

Fetal pleural effusion has been reported to be associated with chromosomal abnormalities, genetic syndromes, obstructive uropathy, lymphatic vessel abnormalities such as Noonan syndrome, RASopathy and congenital lymphatic anomalies, thoracic cavity defects, Rh or ABO incompatibility, non-immune hydrops fetalis, infections, congenital cardiac anomalies, metabolic diseases and hematologic diseases such as α-thalassemia. This review provides a comprehensive view of specific and non-specific chromosome aberrations associated with fetal pleural effusion which is useful for genetic counseling and fetal therapy at prenatal diagnosis of fetal pleural effusion.

Novel premature termination codon in the FOXP3 gene as the cause of familial hydrops fetalis in males.

A 19-year-old, G1P0, pregnant person was referred at 20w2d gestation for evaluation due to non-immune hydrops fetalis (NIHF), which was confirmed at the time of evaluation. Amniocentesis was performed at 20 w4d, and FISH, karyotype, chromosomal microarray, and exome sequencing (ES) were ordered. Trio ES identified a novel hemizygous c.142 C > T (p.Arg48*; maternally inherited) variant in the FOXP3 gene, resulting in a premature termination codon and establishing the diagnosis of immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome. Intrauterine fetal demise (IUFD) was diagnosed at 21w3d. CVS was performed at 12w1d in a subsequent pregnancy (male fetus) and the known familial variant was identified. NIHF was identified at 18w1d. Ultrasound at 19w2d revealed IUFD. This is the first report of this variant in a diagnosis of IPEX syndrome, presenting with NIHF and male fetal demise. Genotype-phenotype correlations are not available in many cases of IPEX syndrome, as the same genotype can be present with variable severity in different individuals. Given the near identical presentations in this family, we anticipate a more severe phenotype with this variant. We propose a novel variant resulting in an early premature termination codon as an explanation for the severe presentation of IPEX syndrome in two successive fetuses in this family.

Functional analysis of a novel splice site variant in the ASAH1 gene.

BACKGROUND: Acid ceramidase (ACDase) deficiency is an ultrarare autosomal recessive lysosomal disorder caused by pathogenic N-acylsphingosine amidohydrolase (ASAH1) variants. It presents with either Farber disease (FD) or spinal muscular atrophy with progressive myoclonic epilepsy (SMA-PME). OBJECTIVE: The study aims to identify a novel splice site variant in a hydrops fetus that causes ASAH1-related disorder, aid genetic counseling, and accurate prenatal diagnosis. METHODS: We report a case of hydrops fetalis with a novel homozygous mutation in ASAH1 inherited from non-consanguineous parents. We performed copy number variation sequencing (CNV-Seq) and whole exome sequencing (WES) on the fetus and family, respectively. Minigene splicing analyses were conducted to confirm the pathogenic variants. RESULTS: WES data revealed a splice site variant of the ASAH1 (c.458-2A>T), which was predicted to affect RNA splicing. Minigene splicing analyses found that the c.458-2A>T variant abolished the canonical splicing of intron 6, thereby activating two cryptic splicing products (c.456_458ins56bp and c.458_503del). CONCLUSIONS: Overall, we identified a novel splice site variant in the mutational spectrum of ASAH1 and its aberrant effect on splicing. These findings highlight the importance of ultrasonic manifestation and family history of fetal hydrops during ASAH1-related disorders and could also aid genetic counseling and accurate prenatal diagnosis. To the best of our knowledge, this is the shortest-lived account of ASAH1-related disorders in utero with severe hydrops fetalis.

Homozygous SPTA1-associated hereditary pyropoikilocytosis presenting as hydrops fetalis.

INTRODUCTION: Hereditary pyropoikilocytosis (HPP) is a heterogeneous inherited disorder of red blood cell (RBC) membrane and cytoskeletal proteins that leads to hemolytic anemia. HPP is characterized by marked poikilocytosis, microspherocytes, RBC fragmentation, and elliptocytes on peripheral blood smear. Mutations in SPTA1 can cause HPP due to a quantitative defect in α-spectrin and can lead to profound fetal anemia and nonimmune hydrops fetalis, which can be managed with intrauterine transfusion. CASE PRESENTATION: We present a case of a 26-year-old G4P2102 woman of Amish-Mennonite ancestry with a pregnancy complicated by fetal homozygosity for an SPTA1 gene variant (SPTA1c.6154delG) as well as severe fetal anemia and hydrops fetalis, which was managed with four intrauterine transfusions between 26 and 30 weeks gestation. Pre-transfusion peripheral smears from fetal blood samples showed RBC morphology consistent with HPP. The neonate had severe hyperbilirubinemia at birth, which has resolved, but remains transfusion-dependent at 6 months of life. DISCUSSION/CONCLUSION: To our knowledge, this is the first report that correlates homozygosity of the SPTA1c.6154delG gene variant with RBC dysmorphology and establishes the diagnosis of HPP.

Deciphering and disrupting PIEZO1-TMEM16F interplay in hereditary xerocytosis.

ABSTRACT: Cell-surface exposure of phosphatidylserine (PS) is essential for phagocytic clearance and blood clotting. Although a calcium-activated phospholipid scramblase (CaPLSase) has long been proposed to mediate PS exposure in red blood cells (RBCs), its identity, activation mechanism, and role in RBC biology and disease remain elusive. Here, we demonstrate that TMEM16F, the long-sought-after RBC CaPLSase, is activated by calcium influx through the mechanosensitive channel PIEZO1 in RBCs. PIEZO1-TMEM16F functional coupling is enhanced in RBCs from individuals with hereditary xerocytosis (HX), an RBC disorder caused by PIEZO1 gain-of-function channelopathy. Enhanced PIEZO1-TMEM16F coupling leads to an increased propensity to expose PS, which may serve as a key risk factor for HX clinical manifestations including anemia, splenomegaly, and postsplenectomy thrombosis. Spider toxin GsMTx-4 and antigout medication benzbromarone inhibit PIEZO1, preventing force-induced echinocytosis, hemolysis, and PS exposure in HX RBCs. Our study thus reveals an activation mechanism of TMEM16F CaPLSase and its pathophysiological function in HX, providing insights into potential treatment.

RASopathies are the most common set of monogenic syndromes identified by exome sequencing for nonimmune hydrops fetalis: A systematic review and meta-analysis.

RASopathies are a group of malformation syndromes known to lead to nonimmune hydrops fetalis (NIHF) in severe presentations. Pathogenic variants can be de novo or parentally inherited. Despite being a known frequent presentation, the fraction of monogenic NIHF cases due to RASopathies is limited in the literature. Also, the specific parental contribution of RASopathies to NIHF is not well described. Our objective was to review pooled exome sequencing (ES) diagnostic yield of RASopathies for NIHF and to determine the parental contribution of RASopathy to NIHF. We performed a systematic review of prenatal ES studies from January 1, 2000 to August 1, 2022. Thirty-six studies met inclusion criteria. Cases with RASopathy gene variants were reviewed. NIHF cases were further classified as isolated or non-isolated. Thirty-six ES studies including 46 pregnancies with NIHF and a diagnosed RASopathy were reviewed. Forty-four diagnostic variants and 2 variants of uncertain significance in 12 RASopathy genes were identified. Expanding on what was previously published, a total of 506 NIHF cases were extracted with 191 cases yielding a positive diagnosis by ES. The overall rate of RASopathy diagnosis in clinically diagnosed NIHF cases was 9% (44/506). The rate of RASopathy diagnosis among NIHF cases with positive genetic diagnosis by ES was 23% (44/191). Of the 46 cases identified, 13 (28%) variants were parentally inherited; specifically, 5/13 (38%) maternal, 3/13 (23%) paternal, 2/13 (15%) biparental, and 3/13 (23%) unspecified. Majority of NIHF cases 29/46 (63%) were isolated. Among NIHF cases with positive ES diagnoses, RASopathy diagnostic yield by ES was 23%. NIHF secondary to RASopathies was parentally inherited in 28% of cases. Most cases of NIHF due to RASopathy were isolated, with no prenatal detection of associated anomalies.

SAM domain variants of EPHB4 associated with aberrant signaling are linked to lymphatic-related fetal hydrops and facial dysmorphology.

Variants in EPHB4 (Ephrin type B receptor 4), a transmembrane tyrosine kinase receptor, have been identified in individuals with various vascular anomalies including Capillary Malformation-Arteriovenous Malformation syndrome 2 and lymphatic-related (non-immune) fetal hydrops (LRHF). Here, we identify two novel variants in EPHB4 that disrupt the SAM domain in two unrelated individuals. Proband 1 presented within the LRHF phenotypic spectrum with hydrops, and proband 2 presented with large nuchal translucency prenatally that spontaneously resolved in addition to dysmorphic features on exam postnatally. These are the first disease associated variants identified that do not disrupt EPHB4 protein expression or tyrosine-kinase activity. We identify that EPHB4 SAM domain disruptions can lead to aberrant downstream signaling, with a loss of the SAM domain resulting in elevated MAPK signaling in proband 1, and a missense variant within the SAM domain resulting in increased cell proliferation in proband 2. This data highlights that a functional SAM domain is required for proper EPHB4 function and vascular development.

Biallelic truncating TTN variants in M-band encoding exons cause a fetal lethal titinopathy.

To report two novel TTN variants associated with fetal recessive titinopathy, thereby broadening the range of TTN variants that can lead to titinopathy. Clinical information on the fetus and parents was gathered, and genomic DNAs were extracted from the fetal tissue and family members' peripheral blood samples. Exome sequencing on fetal DNA was performed and following bioinformatics analysis, the suspected pathogenic variants were confirmed through Sanger sequencing. Prenatal ultrasound performed at 29 weeks of gestation revealed hydrops fetalis, decreased fetal movements, multiple joint contractures and polyhydramnios. Intrauterine fetal death was noted in the third trimester. Exome sequencing revealed compound heterozygous variants in the TTN gene: a paternally inherited allele c.101227C>T (p.Arg33743Ter) and a maternally inherited c.104254C>T (p.Gln34752Ter) allele. These variants have not been previously reported and are evaluated to be likely pathogenic according to the American College of Medical Genetics and Genomics guidelines. We report a fetus with hydrops fetalis and arthrogryposis multiplex congenita associated with a compound heterozygote in the TTN gene. Our report broadens the clinical and genetic spectrum associated with the TTN-related conditions.

Prenatal diagnosis of mucopolysaccharidosis type VII facilitating treatment in neonatal period.

Duo exome testing was performed on a fetus conceived via in vitro fertilization with an egg donor. The fetus presented with non-immune hydrops fetalis (NIHF) at 20 + 0 weeks gestation. Two variants were detected in the GUSB gene. Biallelic pathogenic variants cause mucopolysaccharidosis type VII (MPS-VII), which can present with NIHF prenatally. At the time of analysis and initial report, one variant was classified as likely pathogenic and the other as of uncertain clinical significance. Biochemical testing of the amniotic fluid supernatant showed elevated glycosaminoglycans and low ß-glucuronidase activity consistent with the diagnosis of MPS-VII. This evidence allowed the upgrade of the pathogenicity for both variants, confirming the diagnosis of MPS-VII. The infant was born at 36 + 5 weeks and enzyme replacement therapy (ERT) using vestronidase was initiated at 20 days with planning for hematopoietic stem cell transplant ongoing. The ERT therapy has been well tolerated, with decreasing quantitative urine glycosaminoglycans. Long-term follow up is required to determine whether treatment has been successful. This case demonstrates the utility of alternative testing methods to clarify the pathogenicity of variants and the clinical utility of obtaining a diagnosis antenatally in facilitating treatment in the neonatal period, and specifically highlights MPS-VII as a treatable cause of NIHF.

Resolving fetal hydrops - A rare entity.

Non-immune hydrops fetalis (NIHF) is abnormal accumulation of serous fluid in ≥2 interstitial spaces with no evidence of maternal red cell alloimmunization. Leaving a few treatable conditions, it is generally considered as a sign of poor fetal outcome. Bi-allelic variants in THSD1 have been found to be to be associated with phenotypes ranging from lethal NIHF to persistent edema. Here, we report a family with non-immune hydrops in two successive pregnancies. Whole exome sequencing in second pregnancy identified a homozygous truncating variant in THSD1 (NM_018676:c.892G>T:p.Glu298Ter). Postnatal follow up showed gradual resolution of the accumulated fluid and normal development. This report further strengthens the association of variants in THSD1 with NIHF.

Single-tube multiplex real-time PCR with EvaGreen and high-resolution melting analysis for diagnosis of α0-thalassemia--SEA,--THAI, and--CR type deletions.

Regions with a high prevalence of α-thalassemia (α-thal) require simple, rapid, and accurate tests for carrier screening and prenatal diagnosis. Diagnosis of multiple deletions in a single tube is necessary to clearly identify individuals with α0-thalassemia in the routine setting, especially in at-risk couples. Therefore, we aimed to develop a single-tube multiplex real-time PCR with EvaGreen and high-resolution melting (HRM) analysis for the identification of α0-thalassemia Southeast Asian (SEA), Thai and Chiang Rai (CR) type deletions. The results of the HRM analysis indicated that the amplified fragments from α0-thal--CR,--THAI,--SEA, and the wild-type α-globin gene had specific peak heights at mean melting temperature (Tm) values of 85.40°C, 86.50°C, 87.65°C, and 91.04°C, respectively. The frequencies of α0-thal--SEA,--THAI,--CR obtained from routine testing in 2,135 samples were 17.89%, 0.19% and 0.19%, respectively. This method would be useful for preventing Hb Bart's hydrops fetalis. Detection of multiple deletions in a single run is cost-effective, highly accurate and timesaving. This technique could enable wider α-thalassemia diagnosis in high prevalence areas and served as an example for thalassemia routine setting.

Two unrelated fetuses with ITPR1 missense variants and fetal hydrops.

We describe two fetuses from unrelated families with likely pathogenic variants in ITPR1 that presented with nonimmune fetal hydrops. Trio exome sequencing revealed a de novo heterozygous likely pathogenic missense variant c.7636G > A (p.Val2531Met) in ITPR1 (NM_001378452.1) in proband 1 and a de novo heterozygous likely pathogenic missense variant c.34G > A [p.Gly12Arg] in proband 2. Variants in ITPR1 have been associated with several genetic conditions, including spinocerebellar ataxia 15, spinocerebellar ataxia 29, and Gillespie syndrome. Our report on two patients details a previously undescribed severe fetal presentation of nonimmune hydrops fetalis associated with missense variants in the ITPR1 gene.

[Free sialic acid storage disorders with fetal hydrops in a neonate]. / å”¾æ¶²é ¸è´®ç§¯ç—‡ä¼´èƒŽå„¿æ°´è‚¿1例.

A boy, aged 3 hours, was admitted due to a prenatal diagnosis of fetal hydrops at 3 hours after resuscitation for birth asphyxia. Prenatal examination at 5 months of gestation showed massive ascites in the fetus, and after birth, the boy had the manifestations of systemic hydroderma, massive ascites, coarse face, and hepatomegaly. Genetic testing revealed heterozygous mutations in the SLC17A5 gene, and there was a significant increase in urinary free sialic acid. Placental pathology showed extensive vacuolization in villous stromal cells, Hofbauer cells, cytotrophoblast cells, and syncytiotrophoblast cells in human placental chorionic villi. The boy was finally diagnosed with free sialic acid storage disorders (FSASDs). This is the first case of FSASDs with the initial symptom of fetal hydrops reported in China. The possibility of FSASDs should be considered for cases with non-immune hydrops fetalis, and examinations such as placental pathology and urinary free sialic acid may help with early diagnosis and clinical decision making.

Molecular characterization of a novel 83.9-kb deletion of the α-globin upstream regulatory elements by long-read sequencing.

Inherited deletions of upstream regulatory elements of α-globin genes give rise to α-thalassemia, which is an autosomal recessive monogenic disease. However, conventional thalassemia target diagnosis often fails to identify these rare deletions. Here we reported a family with two previous pregnancies of Hb Bart's hydrops fetalis and was seeking for prenatal diagnosis during the third pregnancy. Both parents had low level of Hemoglobin A2 indicating α-thalassemia. Conventional Gap-PCR and PCR-reverse dot blot showed the father carried -SEA deletion but did not identify any variants in the mother. Multiplex ligation-dependent probe amplification identified a deletion containing two HS-40 probes but could not determine the exact region. Finally, a long-read sequencing (LRS)-based approach directly identified that the exact deletion region was chr16: 48,642-132,584, which was located in the α-globin upstream regulatory elements and named (αα)JM after the Jiangmen city. Gap-PCR and Sanger sequencing confirmed the breakpoint. Both the mother and fetus from the third pregnancy carried heterozygous (αα)JM, and the fetus was normally delivered at gestational age of 39 weeks. This study demonstrated that LRS technology had great advantages over conventional target diagnosis methods for identifying rare thalassemia variants and assisted better carrier screening and prenatal diagnosis of thalassemia.

Diagnosis of α-thalassaemia by colorimetric gap loop mediated isothermal amplification.

α-Thalassaemia is an inherited haemoglobin disorder that results from the defective synthesis of α-globin protein. Couples whom both carry the α-thalassaemia 1 gene are at risk of having a foetus with the most severe thalassaemia, Hb Bart's hydrops fetalis, with a risk of maternal mortality. However, haematological parameters alone cannot distinguish between a α-thalassaemia 1 carrier and a homozygous α-thalassaemia 2, in which one α-globin gene has been deleted on each chromosome. A rapid and accurate molecular detection assay is essential for prevention of the disease in populations where α-thalassaemia 1 is common. Multiplex Gap-PCR analysis is widely used for diagnosis of α-thalassaemia. However, the technique requires a thermocycler and post-amplification processing, which limits its application in primary care or in rural areas in developing countries. Loop mediated isothermal amplification (LAMP) amplifies target DNA at a constant temperature and does not require a thermocycler. This study developed a colorimetric Gap-LAMP using malachite green to allow naked eye visualization of two deletional α-thalassaemia 1 commonly found in Asian populations, the Southeast Asian type (--SEA) and the Thai type (--THAI) deletions. The Gap-LAMP was performed on DNA samples from 410 individuals carrying various α-thalassaemia gene defects with 100% concordance with conventional Gap-PCR analysis. This method eliminates post-amplification processing or the use of expensive sophisticated equipment and allows screening large populations for the prevention and control of α-thalassaemia.

Likely pathogenic variant in the BICD2 gene in fetus presenting with non-immune hydrops.

Trio exome sequencing was performed on a fetus presenting with severe hydrops fetalis at 21 + 0 weeks gestation. A novel de novo BICD2 missense variant was identified in the fetus. Pathogenic variants in the BICD2 gene are associated with lower extremity-predominant spinal muscular atrophy. The variant was initially classified as a variant of uncertain clinical significance (VUS) as at the time of analysis and initial report, pathogenic variants in the BICD2 gene specifically had not been associated with fetal hydrops and no other abnormalities had been detected. It was agreed in multidisciplinary team discussions to include the variant in the report as a VUS recommending phenotypic follow-up. The pregnancy was terminated and post-mortem findings were in keeping with a BICD2-pathogenic variant. In addition, a paper was published reporting another case with a pathogenic BICD2 variant presenting with fetal hydrops. The variant classification was then upgraded to class 4 likely pathogenic and reported as consistent with the diagnosis. This case demonstrates the importance of reporting these new gene/phenotypes in enabling others in the classification of variants, staying up-to-date with literature and following up phenotype for class 3 variants of interest.

Fetal hydrops caused by a novel pathogenic MECOM variant.

We report a fetus with hydrops, congenital heart disease and bilateral radioulnar synostosis caused by a novel pathogenic MECOM variant. The female fetus was referred for post-mortem examination after fetal hydrops and intrauterine death was diagnosed at 20 weeks gestation. Post-mortem examination confirmed fetal hydrops, pallor, truncus arteriosus and bilateral radioulnar synostosis. Trio whole genome sequencing analysis detected a novel de novo heterozygous pathogenic loss-of-function variant in MECOM (NM_004991), associated with a diagnosis of Radioulnar Synostosis with Amegakaryocytic Thrombocytopenia 2 (RUSAT-2). RUSAT-2 is a variable condition associated postnatally with bone marrow failure, radioulnar synostosis and congenital anomalies. RUSAT-2 is not currently associated with a prenatal phenotype or fetal demise, and was not present on diagnostic NHS prenatal gene panels at time of diagnosis. This case highlights the diagnostic value of detailed phenotyping with post-mortem examination, and of using a broad sequencing approach.