Lysosomal storage diseases as a cause of non-immune hydrops fetalis

Non-immune hydrops fetalis (NIHF) is a rare condition that occurs during pregnancy and is characterized by excessive fluid accumulation in two or more fetal body cavities and tissues (ascites, pleural effusion, hydropericardium, and generalized skin edema), excluding alloimmunization [1]. Typically, NIHF is discovered incidentally during routine prenatal examination. The timing, quantity of fluid, and extent of the condition directly influence fetal prognosis, with the underlying cause affecting symptom development. Various causes of NIHF exist, with chromosomal aneuploidies being common in early pregnancy and often identified via increased nuchal translucency thickness and gross malformations in first-trimester prenatal screenings. In the later stages of pregnancy, the primary causes include cardiovascular abnormalities, infections, gastrointestinal and respiratory pathologies, hematological disorders, severe fetal anemia, lymphatic dysplasia, and monogenic diseases [2, 3]. Despite the wide range of causes, the etiology of NIHF remains unknown in a significant number of cases (41–80%) [2, 4].

Advances in genetic diagnostic techniques now allow for comprehensive examination of such patients, facilitating the identification of the causes underlying NIHF.

Among the etiological factors contributing to NIHF are hereditary metabolic diseases, constituting a vast group of over 800 monogenic diseases that are primarily inherited in an autosomal recessive manner. These disorders result from deficiencies in the specific metabolic pathways. Among hereditary metabolic diseases, lysosomal storage diseases (LSDs) form a notable subgroup linked to lysosomal dysfunction. Each LSD, although individually rare, collectively affects approximately 1:5000–1:8000 newborns. LSDs stem from genetic deficiencies in lysosomal hydrolase activity or in the transport system for proteins and substrates into lysosomes. Consequently, macromolecule breakdown or transport is impaired, leading to the excessive accumulation of metabolic products in lysosomes, culminating in cellular dysfunction across organs and tissues. Symptoms of LSDs typically manifest within the first year of life. Rarely, clinical manifestations may occur prenatally, as described in the literature as hepatosplenomegaly, isolated ascites, dysostosis, cardiomyopathy, brain structural changes, polyhydramnios, and NIHF [5–12].

One form of hereditary LSD is mucopolysaccharidosis (MPS), characterized by congenital enzyme deficiencies responsible for glycosaminoglycan degradation. This leads to their accumulation in various body tissues, causing improper bone, cartilage, and connective tissue formation [13]. Consequently, MPS affects the skeletal, nervous, respiratory, cardiovascular, gastrointestinal, ocular, and auditory systems [5]. Seven types of MPS syndromes with 14 subgroups have been identified to date [15]. MPS, excluding type II (Hunter syndrome) with an X-linked inheritance, follows an autosomal recessive pattern. The overall incidence of MPS is approximately one in 25,000 live births [16]. The incidence rates vary according to MPS subtype. Symptom onset in MPS correlates with genotype and encompasses systemic manifestations, such as growth restriction, cognitive impairment, musculoskeletal abnormalities, cardiac valve issues, coronary artery disease, gastrointestinal problems, hepatosplenomegaly, umbilical hernias, sensorineural hearing loss, upper airway blockages, and visual impairment [16]. Clinical presentations range from mild forms with normal life expectancy to severe phenotypes resulting in early infancy or intrauterine death [5, 13].

Another example of LSD is galactosialidosis, characterized by a secondary deficiency of neuraminidase-1 and beta-galactosidase. This leads to the intracellular accumulation of gangliosides, sialic-containing glycolipids, and glycoproteins, resulting in abnormal elastic fiber formation, structural changes, myelination alterations, and varying degrees of dysostosis. The disease presents as three types: early infantile, late infantile, and adolescent/adult, exhibiting coarse facial features, cognitive impairment, ataxia, and sensorineural hearing loss across all types. Antenatal manifestations of galactosialidosis include hepatosplenomegaly, cardiomyopathy, multiple dysostoses, and frequently, NIHF. The exact prevalence of galactosialidosis remains unknown, with approximately 150 cases reported in the literature and no published epidemiological data for the Russian Federation [17].

We present two clinical cases illustrating the antenatal diagnosis of LSDs: MPS VII type (Sly syndrome) identified in a fetus with NIHF at 21–22 weeks of pregnancy and galactosialidosis diagnosed at delivery.

Materials and methods

The patients sought medical attention at the 21st and 25th week of pregnancy. According to the algorithm developed by Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of the Russian Federation (hereinafter referred to as the Center), they underwent an expert ultrasound examination (US), Doppler study with measurement of the mean blood flow velocity in the fetal middle cerebral artery, fetal echocardiography (ECHO-CG), and genetic counseling [18–20].

Clinical evaluation also included transabdominal amniocentesis, analysis of amniotic fluid in fetuses with NIHF for infectious screening, and DNA extraction for subsequent molecular karyotyping and whole-exome trio sequencing. The genetic part of the study was conducted at the Institute of Reproductive Genetics of the Center. Infectious screening included testing of amniotic fluid using polymerase chain reaction (PCR) for cytomegalovirus, Epstein–Barr virus, herpes simplex types 1 and 2, parvovirus B19, and bacteriological culture of amniotic fluid. Peripheral blood samples were collected from parents, and written informed consent was obtained for whole-exome trio sequencing. DNA was isolated from amniotic fluid using the IGENatal kit (Spain), and from blood cells using a kit of reagents for DNA isolation PROBA-MC MAX (Russia). Patient DNA sequencing was performed using the NovaSeq 6000 platform (WES, whole exome sequencing). The IDT XGen Exome Hyb Panel v2 enrichment kit was used to enrich target fragments. Sequencing data were processed using an algorithm that included alignment of reads to the reference genome sequence hg38 and collating and filtering of variants by quality. For all variants that passed quality filtering, annotation was carried out using the Ensembl Variant Effect Predictor (VEP), and a number of algorithms were used to predict the significance of variants (SIFT, PolyPhen-2, SpliceAI). Assessment of the clinical significance (pathogenicity) of the identified variants was carried out based on the ACMG recommendations [21] and Russian recommendations for the interpretation of data [22] obtained by high-throughput sequencing [23–25].

The study was reviewed and approved by the Research Ethics Committee of the Academician V.I. Kulakov National Medical Research Center for Obstetrics, Gynecology and Perinatology, Ministry of Health of the Russian Federation.

Results

Case 1. A 29-year-old pregnant woman, Z.O., who had her second pregnancy, presented at the Center at 20 weeks due to NIHF. The first pregnancy ended at 21 weeks after termination due to NIHF. This was the patient’s second pregnancy that occurred spontaneously. At 13 weeks, according to the conclusion of prenatal screening in the first trimester, low risks of chromosomal abnormalities were identified (risk for trisomy 21 – 1 in 7802, trisomy 18 – <1 in 20,000, trisomy 13 – <1 in 20,000), and no congenital malformations were identified. Repeat ultrasonography was performed at 18 weeks and 4 days. Subcutaneous fetal edema was noted: up to 1.8 mm along the anterior wall, 7.5 mm along the posterior wall, ascites 8.5 mm, severe hepatomegaly and splenomegaly. An effusion of 7.1 mm thickness was detected in the pleural cavity and an effusion of 7.1 mm in the pericardium. The amount of amniotic fluid was within normal limits, the placenta was thickened to 32 mm, and the structure did not change. When performing an expert ultrasound at the Center at 20 weeks 2 days, anasarca was diagnosed; in the projection of the torso, there was thickness 7.4 mm, also swelling in the projection of the lower extremities, bilateral hydrothorax, ascites, the amount of ascitic fluid was 16 mm, and swelling of the subcutaneous tissue of the neck. An ECHO-CG of the fetus was performed; fluid in the pericardium was 1 mm, and no evidence of gross congenital heart disease was detected in the fetus. Medical and genetic counseling was performed. Considering the repeated case of NIHF in this married couple and the persistent desire of the parents to prolong pregnancy, a decision was made to conduct invasive prenatal diagnostics. At 20 weeks and 4 days, transabdominal amniocentesis was performed, and the amniotic fluid was sent for infectious and molecular genetic studies. According to infectious screening data, cytomegalovirus was diagnosed in the amniotic fluid using PCR; IgG antibodies to the coronavirus SARS-CoV-2 1269 BAU/ml (positive) and IgG antibodies to the Epstein–Barr virus were detected. According to prenatal molecular karyotyping data on DNA microarrays (CytoScan), the analyzed sample contained DNA with a male genotype, and no aneuploidy was detected. Due to the lack of data on chromosomal pathology in a living fetus, parental and fetal material was sent for high-throughput sequencing (whole-exome trio sequencing). Considering the presumed infectious genesis of NIHF, immunoglobulin therapy 5000 mg intravenously was administered intravenously in a hospital setting.

During a follow-up examination 10 days later, negative dynamics were observed: the amount of ascitic fluid was 25 mm, pronounced swelling of the subcutaneous fat, and bilateral hydrothorax (on the left, 10 ml; on the right, 11 ml). According to the maximum blood flow velocity in the middle cerebral artery, the fetus is likely to develop severe anemia (1.8 MoM). Oligohydramnios and placental thickening were also observed. Severe anemia in the fetus is regarded as a manifestation of an infectious process; therefore, transabdominal amniocentesis, evacuation of ascitic fluid from the fetus, intraperitoneal transfusion of washed red blood cells according to individual selection in a volume of 20.0 ml, and intraperitoneal administration of 2 mg immunoglobulin were performed. During a follow-up examination one day later, according to the maximum blood flow velocity in the middle cerebral artery, the fetus showed positive dynamics. Despite therapy, antenatal fetal death was diagnosed at 22 weeks and 6 days of gestation, and the patient delivered vaginally.

Pathological examination of the fetus confirmed prenatally detected anomalies. The male fetus had characteristic signs of dropsy, morphologically manifested as anasarca, ascites, bilateral hydrothorax, severe cerebral edema, maceration of the skin (more than 50%), and total pulmonary atelectasis. According to anthropometry, the fetus was 23 weeks old. When examining the placenta, it was large for the gestational age, the predominance of angiogenesis with vessel branching, and excessive tortuosity of the umbilical cord (0.09).

According to whole-exome trio sequencing data, likely pathogenic variants related to the phenotype (according to the ACMG gene list) were detected in the fetus. A previously undescribed heterozygous variant of the nucleotide sequence in the GUSB gene (7-65961051-ACT-A) was identified, leading to a reading frame shift and impaired synthesis of the full-length protein (p.Arg600fs, NM_000181), inherited from the mother. In the same GUSB gene, a previously undescribed heterozygous variant of the nucleotide sequence (7-65974700-C-A) was identified, which led to an amino acid substitution at position 357 of the protein (p.Arg357Leu, NM_000181) inherited from the father.

Case 2. This clinical observation represents galactosialidosis in the fetus at the time of delivery and follow-up history of the newborn.

A 33-year-old pregnant woman K.V. presented for a prenatal consultation at 25 weeks of gestation. The first pregnancy ended in the termination of pregnancy at 5 weeks at the place of residence at the request of the patient. This was her second spontaneous pregnancy. At 11 weeks, the patient had acute respiratory viral infection with low-grade fever and herpetic rashes on the face. The patient received symptomatic therapy. At 13 weeks, according to prenatal screening in the first trimester, low risks of chromosomal abnormalities were identified, and no congenital malformations were detected. A repeat ultrasound was performed at 19 weeks, including ventriculomegaly, which detected agenesis of the corpus callosum in the fetus. After a prenatal consultation in Kirov, considering the persistent desire of the parents, it was decided to prolong this pregnancy. Subsequently, when performing ultrasound at 24 weeks, NIHF was first diagnosed with mild ascites, hepatomegaly, and ventriculomegaly. An examination at the Center confirmed the fetal pathology. According to the ECHO-KG of the fetus, no evidence of gross congenital heart disease was identified. Transabdominal amniocentesis was then performed. Amniotic fluid was sent for infectious screening and genetic testing. Infectious screening using PCR and bacteriological culture revealed no evidence of infection. Due to the lack of data on chromosomal pathology in a living fetus, parental and fetal material was sent for high-throughput sequencing (whole-exome trio sequencing).

During the prenatal consultation at 28 weeks of pregnancy, negative dynamics were noted. Ultrasound findings included ascites (25 mm), hydropericardium, hepatomegaly, minor ventriculomegaly (10–12 mm), bilateral hydroceles, and soft tissue edema.  Doppler ultrasound findings revealed mild fetal anemia (1.3 MoM). According to the ECHO-CG, moderate cardiomegaly, right ventricular hypertrophy, atrioventricular regurgitation, and systole-diastolic dysfunction were observed. Further observation at the place of residence and repeated consultations at 30–32 and 34–36 weeks of pregnancy were recommended. According to the conclusion of the prenatal consultation, delivery was planned at the Center; however, at 36 weeks of pregnancy, owing to the onset of regular labor, preterm spontaneous birth occurred in the city of Kirov. A live preterm infant was born with a birth weight of 3620 g, length of 53 cm, Apgar score of 7/8, and severe dropsy symptoms. By the time of birth, sequencing data for the complete whole-exome trio sequencing were obtained; a previously undescribed heterozygous variant of the nucleotide sequence in the CTSA gene (20-45891990-C-T) was found in the fetus, leading to an amino acid substitution at position 90 of the protein (p.Ser90Leu, NM_000308, rs137854542), inherited from the father. In the same CTSA gene, a previously undescribed heterozygous variant of the nucleotide sequence (20-45894902-G-A) was identified, leading to disruption of the canonical splicing site (c.948+1G>A, NM_000308) inherited from the mother. Homozygous and compound heterozygous variants of this gene are associated with a monogenic autosomal recessive disease in the LSD group, galactosialidosis. After birth, the child was observed in the neonatal intensive care unit and was on mechanical ventilation for respiratory failure. A puncture of the scrotum was performed, 170 ml of serous fluid was evacuated, the anterior abdominal wall was punctured, and ascitic fluid was evacuated. The child received symptomatic therapy (cardiotonic, diuretic, antibacterial, or antimycotic). During hospitalization, the child was examined by specialists in various fields (surgeons, pulmonologists, ophthalmologists, pediatric cardiologists, neurologists, gastroenterologists, and geneticists) and sensorineural hearing loss was diagnosed. During the 1.5 months of observation and treatment, the child’s condition showed positive dynamics with a decrease in soft tissue edema, ascites, resolution of pulmonary edema, and hydrocele. The child was discharged and sent for consultation on the treatment of galactosialidosis at the Veltischev Research and Clinical Institute for Pediatrics. At the age of 8 months, the child was diagnosed with a new coronavirus infection, which manifested as fever (38.5°C), lethargy, and intestinal disorders. Within one month, the child's condition worsened. Examination revealed bilateral pneumonia, virus-associated thrombocytopenia, severe anemia, reactive hepatitis, and ascites. Symptomatic and etiotropic therapy and transfusion of washed erythrocytes were administered. Due to the severity of the condition and the progression of the underlying disease, the child was placed in palliative status and transferred to a specialized facility for inpatient palliative care. The child died at 10 months of age.

Discussion

When performing molecular karyotyping using amniotic fluid DNA, no aneuploidies or significant microdeletions/microduplications were identified in both cases. Therefore, chromosomal abnormalities were excluded as a cause of NIHF.

In the first clinical example, according to the trio-whole exome sequencing data, two variants were found in the GUSB gene in a compound heterozygous state, and the identified variants were validated using Sanger sequencing.

Thus, the disease diagnosed in the fetus during the first clinical observation belongs to one of the LSDs, mucopolysaccharidosis type VII.

According to the literature, MPS VII type (Sly syndrome) is a heterogeneous progressive metabolic disorder caused by pathogenic and likely pathogenic variants in the GUSB gene (chromosome 7q11), which leads to deficiency of the lysosomal enzyme β-glucuronidase and subsequent accumulation of glycosaminoglycans: chondroitin sulfate (CS), dermatan sulfate (DS) and heparan sulfate (HS) in lysosomes, causing multiple organ dysfunction [9, 26, 27].

MPS type VII is an extremely rare disease, with an estimated prevalence of less than 1:1,000,000; however, accurate assessment is difficult due to the lack of accurate epidemiological data, which is associated with difficulties in diagnosing this disease. The extremely low incidence and diagnosis of MPS type VII indicates that literature data are limited. MPS type VII was first described in 1973 by Dr. William Sly, and many other cases were subsequently identified in patients from all continents [28].

The phenotype of MPS type VII is heterogeneous and varies from severe forms – antenatal fetal death, manifesting mainly as NIHF, to milder forms with late onset of manifestation and high survival to adulthood with normal or almost normal intelligence. According to the literature, this type of MPS can manifest itself at the antenatal stage and has severe manifestations, such as NIHF [7, 9, 29–31]. In this clinical observation, there were ultrasound signs of MPS in the fetus: polyhydramnios, generalized subcutaneous edema, ascites, bilateral hydrothorax, hydropericardium, and cardiomyopathy, which correspond to the literature describing antenatal cases of this disease [5–9]. MPS type VII, with early manifestation at the antenatal stage, has an extremely unfavorable prognosis and often results in fetal death. Onset in infancy or childhood is associated with low life expectancy, although with a milder phenotype, life expectancy may increase [32, 33]. In general, patients with MPS type VII have short stature and progressive skeletal deformities, leading to spinal curvature, long bone deformity, macrocephaly, hip dysplasia, chest deformity, facial dysmorphism, and hepatosplenomegaly. The main manifestations of damage to the nervous system for this syndrome are mental retardation, sensorineural hearing loss, and eye damage are also common signs [9, 27].

The main treatments for MPS are specific enzyme replacement therapy (for MPS type VII, the enzyme Westronidase alfa, which is a formulation of recombinant human β-glucuronidase approved in 2017 by the Food and Drug Administration (FDA)) and bone marrow transplantation, which increases the duration and quality of life of patients [5, 34]. In 2021, studies on intrauterine fetal enzyme replacement therapy began as part of phase I clinical trials on experimental animals [7].

In the second clinical observation, according to the data of whole-exome trio sequencing, two variants were found in the CTSA gene in a compound heterozygous state, and based on the data obtained, the fetus was diagnosed with one of the LSDs, galactosialidosis. The patient’s parents were carriers of the probable pathogenic variants described above in a heterozygous state. The identified variants were validated by Sanger sequencing.

Galactosialidosis is an LSD associated with pathogenic and probable pathogenic variants of the CTSA gene, which encodes a glycoprotein associated with the lysosomal enzymes beta-galactosidase and neuraminidase. Loss of protein function leads to a secondary deficiency of these enzymes, which are the main biochemical markers of this disease. Galactosialidosis has a wide range of clinical manifestations. The most severe type of galactosialidosis is early infantile galactosialidosis, and its clinical manifestations at the antenatal stage include non-immune hydrops fetalis (NIHF), hepatosplenomegaly, ventriculomegaly, and various degrees of dysplasia, which can lead to antenatal fetal death or the birth of a child with severe lesions and a characteristic phenotype for this disease. In milder cases, patients experience delayed psychomotor development, the formation of coarse facial features, multiple dysostoses, neurological disorders, myoclonus, ataxia, and impaired pulmonary function, manifested by emphysema associated with a defect in the formation of elastic fibers [17, 35, 36]. Among the causes of NIHF, LSD accounts for 5.3–29% according to various literature data, of which galactosialidosis accounts for 6–28.2% [37, 38].

In this clinical observation, ultrasound signs of NIHF were encountered, accompanied by changes in the internal organs (ascites, hepatosplenomegaly, ventriculomegaly (10–12 mm), and soft tissue edema). These changes are nonspecific and require extensive prenatal examination. Infectious factors were not confirmed during the examination, which necessitated further diagnostic investigation. At birth, children’s symptoms of dropsy were confirmed using instrumental examination methods. As of 2023, no specific treatment has been developed for galactosialidosis. All patients required long-term observation by a neurologist, ophthalmologist, cardiologist, otolaryngologist, and orthopedic traumatologist. Therapy is symptomatic.

The development of modern methods of fetal ultrasound, as well as other technologies for prenatal diagnostics, such as prenatal molecular karyotyping on DNA microarrays, high-throughput sequencing (whole exome “trio” sequencing), and Sanger sequencing in combination with a detailed pathological examination, presents new opportunities for identifying the causes of NIHF. Thus, in the present study, screening ultrasound allowed for the identification of NIHF at 20 weeks of pregnancy, and molecular genetic examination allowed the establishment of a direct cause of lethal fetal pathology.

Genetic counseling in these families is based on information about the high risk of disease recurrence in subsequent pregnancies and the possibility of prenatal (preimplantation and/or prenatal) diagnosis. If the next pregnancy occurs spontaneously in this family, there is a high risk of repeating an unfavorable outcome in 25% of the cases. Considering the autosomal recessive inheritance of these diseases and the heterozygosity of the variants in the mother and father, in 25% of cases, the fetuses will not inherit these pathogenic variants; in 50% of cases, they will be carriers of one of the pathogenic variants and will not have clinical manifestations of one of the LSDs. In the case of a spontaneous pregnancy, it is possible to obtain reliable information about the presence or absence of these likely pathogenic variants in fetuses in the first trimester of pregnancy. At the pregnancy planning stage, it is possible to conduct IVF programs with preimplantation genetic testing of the above-mentioned monogenic diseases (PGT-M) of embryos, with subsequent embryo selection.

Whole-exome sequencing allowed us to expand our understanding of the etiology and pathogenesis of NIHF. An important feature of “trio” whole-exome sequencing is the rapid interpretation of the results, which provides the opportunity to change pregnancy management tactics and opens up opportunities for specific treatment of the fetus or newborn. In real-world practice, prenatal diagnosis of LSD is usually carried out for high-risk families as well as in situations where the disease is particularly severe and does not respond to generally accepted methods of therapy [39]. Diagnosis of this disease in the fetus in connection with an incidental finding during routine ultrasound in the second trimester is of particular interest, emphasizing the importance of identifying the cause in each case of NIHF.

Conclusion

Based on the present study, it can be concluded that patients with non-immune hydrops fetalis (NIHF) show heterogeneity and unique characteristics. Therefore, it is essential to investigate the role of the genetic pathology in each individual case. Owing to the high mortality rate associated with NIHF, advancements in genetic diagnostic methods are crucial to understand the underlying causes of this condition. This, in turn, facilitates the optimization of pregnancy management strategies.

By examining the parents of affected couples, it is possible to predict the risk of future pregnancies being affected by the identified pathology. This enables the expansion of genetic counseling options for couples who have experienced adverse pregnancy outcomes due to NIHF. It also helps to determine the potential risks for future offspring, providing the family with the necessary information to make informed reproductive choices.