Novel IARS1 variants cause syndromic developmental disorder with epilepsy in a Chinese patient and the literature review.

BACKGROUND: Isoleucinyl-tRNA synthetase (IARS) is encoded by the IARS1 gene and catalyzes the binding of isoleucine to specific tRNA. OBJECTIVE: This study aims to investigate the pathogenicity of novel IARS1 variants and the genotype-phenotype association, in order to expand the spectrum of pathogenic variants and phenotypes of IARS1-related disease and provide new evidence for the phenotypic spectrum of IARS1 variants. METHODS: Clinical data of the proband were collected, and trio whole-exome sequencing (WES) was performed on the proband and the parents. Candidate variants were validated using Sanger sequencing. Bioinformatics software was utilized to analyze the functional consequences of identified variants and predict their potential deleteriousness. RESULTS: A 17-month-old female patient presented with microcephaly, left external ear malformation, decreased muscle strength and tone in all limbs, epileptic seizures, global developmental delay, and developmental regression. Trio WES identified compound heterozygous variants in the IARS1 gene, c.120-1G>A and c.2164C>A, which were novel pathogenic and likely pathogenic variants, respectively. The phenotype of developmental regression has not been reported before. Only one patient with IARS1 compound heterozygous variants has been reported in the world to have an epileptic phenotype, and this is the second patient with an epileptic phenotype. Bioinformatics analysis revealed that the splicing variant disrupted the canonical splice donor site, while the missense variant altered the local electrostatics of the IARS1 protein surface, potentially leading to functional abnormalities. CONCLUSION: This study identified novel IARS1 variants and the phenotype of developmental regression, expanding the spectrum of pathogenic variants and phenotypes of IARS1-related diseases and providing new evidence for the rare phenotype of epileptic seizures caused by IARS1 variants.

[Genetic analysis of a child with Complex cortical dysplasia with other brain malformations type 6 due to a p.M73V variant of TUBB gene].

OBJECTIVE: To explore the genetic basis for a child with multiple malformations. METHODS: A child who had presented at Shanxi Provincial Children's Hospital in February 2021 was selected as the study subject. Clinical data of the patient was collected, and whole exome sequencing (WES) was carried out to screen pathogenic variants associated with the phenotype. Candidate variant was validated by Sanger sequencing of her family members. RESULTS: The child had normal skin, but right ear defect, hemivertebral deformity, ventricular septal defect, arterial duct and patent foramen ovale, and separation of collecting system of the left kidney. Cranial MRI showed irregular enlargement of bilateral ventricles and widening of the distance between the cerebral cortex and temporal meninges. Genetic testing revealed that she has harbored a heterozygous variant of NM_178014.4: c.217A>G (p.Met73Val) in the TUBB gene, which was unreported previously and predicted to be likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics (ACMG). The child was diagnosed with Complex cortical dysplasia with other brain malformations 6 (CDCBM6). CONCLUSION: CDCBM is a rare and serious disease with great genetic heterogeneity, and CDCBM6 caused by mutations of the TUBB gene is even rarer. Above finding has enriched the variant and phenotypic spectrum of the TUBB gene, and provided important reference for summarizing the genotype-phenotype correlation of the CDCBM6.

Correlation of TGF-ß signaling pathway gene polymorphisms with unexplained recurrent spontaneous abortion.

BACKGROUND: The association of key genes in the transforming growth factor-ß (TGF-ß) signaling pathway and their gene polymorphisms with unexplained recurrent spontaneous abortion (URSA) is unclear. OBJECTIVE: To investigate the association of gene polymorphisms related to the TGF-ß signaling pathway in URSA women. METHODS: The study population consisted of 80 women with URSA and 90 normal control women, of which 10 women with URSA and 10 normal control women underwent high-throughput sequencing to select loci, and the remaining 70 women with URSA and 80 normal control women underwent flight mass spectrometry experiments to verify gene loci polymorphism. A total of 7 polymorphic loci in interleukin-6 (IL-6), TGF-ß1, TNF-α, SMAD1, and TNFRSF4 genes were screened by high-throughput sequencing combined with a review of databases. An SNP flight mass spectrometer (Mass ARRAY detection system) was applied to detect the polymorphisms and their frequencies in 70 women with URSA and 80 normal control women at the 7 gene loci. RESULTS: Among the 7 loci of IL-6, TGF-ß1, TNF-α, SMAD1, and TNFRSF4 genes, 2 loci were found to have significantly different allele and genotype frequency distributions between the 70 URSA and 80 normal controls, one was the IL-6 gene -174G/C locus (rs1800795), the risk of disease was 2.636 and 3.231 times higher in individuals carrying the C allele and CC genotype than in those carrying the G allele and GG genotype, respectively; the other was the TGF-ß1 gene -509T/C locus (rs1800469), and the risk of disease was 1.959 and 3.609 times higher in individuals carrying the T allele and TT genotype than in those carrying the C allele and CC genotype, respectively. The remaining 5 genetic loci have no statistically significant. CONCLUSION: IL-6 gene -174G/C locus (rs1800795) genotype CC and allele C may be the causative factor of URSA, TGF-ß1 gene -509T/C locus (rs1800469) genotype TT and allele T may be the causative factor of URSA, and polymorphisms of the 2 loci may be associated with URSA.

Prenatal diagnosis in a fetuses with a clenched hands, overlapping fingers, and clubfoot due to MED12 deficiency in three affected siblings: A case report.

A fetal clenched hand with overlapping fingers is more common in aneuploidy syndrome and was not well-documented in MED12 deficiency. This study reports the clinical and genetic findings of three affected siblings from a Chinese family. The chromosome karyotype analysis diagram shows that karyotypes of the three children were normal. Trio whole-exome sequencing and Sanger sequencing verification found that there was a MED12 R296Q variant in normal mothers and their two offspring. A pattern of clenched hand with overlapping fingers (clinodactyly) and clubfoot was found in all the three affected siblings by three-dimensional ultrasound. The discovery of this case shows that even if the chromosome karyotype is normal, comprehensive prenatal genetic diagnosis is required when the ultrasound results show a clenched hand with clinodactyly and clubfoot symptoms.

Low Expression of LEFTY1 in Placental Villi Is Associated with Early Unexplained Miscarriage.

OBJECTIVE: To investigate the function and underlying mechanism of transforming growth factor­beta (TGF-ß)/bone morphogenetic protein (BMP) signaling pathway in early unexplained miscarriage. STUDY DESIGN: Expression profiles of genes involved in TGF-ß/BMP signaling pathway were compared between placental villous tissue samples from 2 women with missed abortion and those from 2 women with induced abortion by microarray assay. The protein expression level of the most downregulated gene­LEFTY1­was further measured using western blotting in another 8 women with missed abortion and 7 women with induced abortion. RESULTS: A total of 24 genes showed differential expression level between the 2 groups. Their functions were further investigated, of which 6 of 13 upregulated genes were TGF-ß responsive genes. The most reduced gene is LEFTY1, an antagonist of TGF-ß ligand. The protein expression level of LEFTY1 was confirmed to show the same trend as microarray using western blotting. CONCLUSION: A reduced expression of LEFTY1 in women with missed abortion was identified as com-pared with women with induced abortion, which may result in a dysregulation of TGF-ß signaling and may be the underlying mechanism of missed abortion.

[Phenotypic and genetic analysis of a child featuring multiple malformations due to copy number variation on chromosome 5].

OBJECTIVE: To determine the origin of chromosomal aberration for a child featuring multiple malformation, and to correlate the genotype with phenotype. METHODS: Routine G-banding was performed to analyze the karyotype of the patient and her parents, and array comparative genomic hybridization (array CGH) was used for fine mapping of the aberrant region. RESULTS: The karyotype of the child was ascertained as 46,XY. Array CGH has mapped a 14.21 Mb deletion to 5p15.2p15.33, and a very small 3.67 Mb duplication to 5q35.3. The patient has presented features such as mental retardation, heart defect, low-set ears, hypertelorism and down-slanting palpebral fissures. CONCLUSION: Chromosome 5 copy number variation can cause multiple malformation. In contrast to routine karyotype analysis, array CGH can map aberrant region with much higher resolution and accuracy.

A case of agonadism associated with y-chromosome rearrangement: cytogenetic and molecular studies.

Testicular regression syndrome (MIM273250) is characterized primarily by absence of gonads in a person of XY karyotype. Phenotypes range from complete female external genitalia (primary or "true" agonadism) to male phenotype with anorchia (testicular regression). Phenotypic differences depend on the stage of embryo development during which testes degenerate. No conclusive mapping can be concluded for the phenotype. We describe a novel case of primary agonadism with a karyotype of 46,X,der(Y)(pter-->q11.23::pter-->p11.31 or p11.2:). Transcriptional analysis revealed little expression of USP9Y and UTY genes on the Y chromosome in our case, which would explain her phenotypes of agonadism with short stature.

A novel splice site mutation in the dentin sialophosphoprotein gene in a Chinese family with dentinogenesis imperfecta type II.

Twenty-four individuals were investigated that spanned six generations in a Chinese family affected with an apparently autosomal dominant form of dentinogenesis imperfecta type II (DGI-II, OMIM #125490). All affected individuals presented with typical, clinical and radiographic features of DGI-II, but without bilateral progressive high-frequency sensorineural hearing loss. To investigate the mutated molecule, a positional candidate approach was used to determine the mutated gene in this family. Genomic DNA was obtained from 24 affected individuals, 18 unaffected relatives of the family and 50 controls. Haplotype analysis was performed using leukocyte DNA for 6 short tandem repeat (STR) markers present in chromosome 4 (D4S1534, GATA62A11, DSPP, DMP1, SPP1 and D4S1563). In the critical region between D4S1534 and DMP1, the dentin sialophosphoprotein (DSPP) gene (OMIM *125485) was considered as the strongest candidate gene. The first four exons and exon/intron boundaries of the gene were analyzed using DNA from 24 affected individuals and 18 unaffected relatives of the same family. DNA sequencing revealed a heterozygous deletion mutation in intron 2 (at positions -3 to -25), which resulted in a frameshift mutation, that changed the acceptor site sequence from CAG to AAG (IVS2-3C-->A) and may also have disrupted the branch point consensus sequence in intron 2. The mutation was found in the 24 affected individuals, but not in the 18 unaffected relatives and 50 controls. The deletion was identified by allele-specific sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis. We conclude that the heterozygous deletion mutation contributed to the pathogenesis of DGI-II.

Rapid molecular prenatal diagnosis of spondyloepiphyseal dysplasia congenita by PCR-SSP assay.

Heterozygous mutations of COL2A1 gene are responsible for type II collagenopathies. The common skeletal phenotypes include achondrogenesis type II, hypochondrogenesis, Stickler dysplasia, Kniest dysplasia, late onset spondyloepiphyseal dysplasia, and spondyloepiphyseal dysplasia congenita (SEDC). Prevention of SEDC can be achieved by prenatal diagnosis. This study reports the first rapid molecular prenatal diagnosis of SEDC performed in China by polymerase chain reaction sequence-specific primer (PCR-SSP) analysis. The pregnant woman we previously reported with SEDC carried the G to A substitution at nucleotide 1510 in exon 23 of COL2A1 gene, which caused a change from glycine to serine at codon 504 (G504S). By the time the woman got pregnant again, she had terminated two pregnancies and still had no child. In the first pregnancy, the molecular mutation of the family was not yet identified, and therefore prenatal diagnosis was unable to be performed by DNA analysis. In the second pregnancy, G504S mutation was found from fetal DNA. At the time of her third pregnancy, the woman and her husband became extremely worried about the potential SEDC for the fetus. For this reason, a quick and reliable molecular prenatal diagnosis of SEDC was performed by a PCR-SSP on an amniocyte sample collected at the 14th week of pregnancy. No mutation of the fetal DNA was identified. The result was obtained within 24 h after the sample was collected. The technique could be applied in confirmatory diagnosis and prenatal diagnosis for the affected family.

[Observation of spermatogenic cells for infertile patients with Y-chromosomal microdeletion].

OBJECTIVE: To assess the spermatogenic function of the infertile patients with Y-chromosomal microdeletion. METHODS: Thirty-five 23-44 years old patients with microdeletions of Y chromosome were included in this study. Three semen analyses confirmed that 26 cases were non-obstructive azoospermia and 9 oligospermia with sperm count < 1 x 10(6)/ml. They were divided into 3 groups by the locus of deletion, 5 cases of AZFa + b + c deletion in group 1, 4 cases of AZFb + c and 3 cases of AZFb deletion in group 2, and 23 cases of AZFc deletion in group 3. Semen was collected and centrifuged, the supernatant removed and the centrifugate applied on the clean slides after dilution. Following Wright's-Giemsa staining, the slides were viewed under the microscope. Testis histopathological biopsy was performed for 6 of the cases. RESULTS: In group 1, no spermatogenic cells were observed but only Sertoli cells in 1 case, with a consistency between the result of spermatogenic cell test and that of testis biopsy. In group 2, spermatogenic cell tests revealed spermatocytes in 6 cases, 2 were proved by testis biopsy with sperm maturation arrest in the primary spermatocyte stage, and spermatogenic cells of all developmental stages were seen in 1 AZFb deletion patient with the same sperm maturation arrest as the above two. In group 3, only primary spermatocytes were detected by spermatogenic cell test in 5 oligospermia patients but no spermatogenic cells in the 15 azoospermia cases, and biopsy revealed 2 cases of Sertoli cell-only syndrome. CONCLUSION: The spermatogenic cell test can effectively assess the spermatogenic function of AZF deletion patients. Non-invasive and easily accepted by patients, it is highly recommendable for the evaluation of spermatogenesis of patients with Y-chromosomal microdeletion.

[A girl with partial monosomy 18q21: cytogenetic and molecular genetics studies].

This study is about a girl with chromosome deletion of 18q and with mental retardation and mild delay of physical development. Based on karyotyping of high resolution, fluorescence in situ hybridization (FISH) and microsatellite analysis mapping to 18q, we found that the patient's karyotype was interpreted as 46,XX,del(18).(pter-->q21:), ish del(18)(D18Z1+,qter-). Detection of D18S979 showed that the region from 18q21.1 to 18qter was deleted, which was originated from her father. There were MBP gene and GALNR gene in the deleted interval in which both of them were lost. In conclusion, deletion of 18q21-->qter including the MBP gene and GALNR gene should be responsible for her mental retardation and mild delay of development.

A novel RNA-splicing mutation in COL1A1 gene causing osteogenesis imperfecta type I in a Chinese family.

BACKGROUND: Osteogenesis imperfecta (OI), also known as brittle bone disease, is a rare heterogeneous group of inherited disorders characterized by low bone mass and increased bone fragility. The four major clinical criteria for diagnosis of OI are osteoporosis with abnormal fragility of the skeleton, blue sclera, dentinogenesis imperfecta, and premature otosclerosis. The presence of two of these abnormalities confirms the diagnosis. More than 90% patients have autosomal dominant mutations in one of the two genes, COL1A1 and COL1A2, that encode the alpha chains of type I collagen. While the diagnosis of OI is still based on clinical and radiological grounds, there is a growing demand for the molecular characterization of causative mutations. Although there have been several studies on the mutational spectra of COL1A1 and/or COL1A2 in Western populations, very few cases have been reported from Asia. The purpose of this study is to report two patients with OI type I in a Chinese family, who had a novel RNA-splicing mutation in COL1A1 gene and describe the molecular, radiological and clinical findings. METHODS: The proband, (case II-5), a 32-y-old Chinese male, and his 7-y-old daughter were diagnosed as OI type I according to their clinical and radiological features. Genomic DNA was extracted from their blood samples and all promoters, exons and exon/intron boundaries of COL1A1 and COL1A2 genes were sequenced. Polymerase chain reaction sequence-specific primers (PCR-SSP) was used to confirm patients' heterozygous state. RESULTS: Direct DNA sequencing analysis of COL1A1 gene revealed a splicing mutation (c.1875+1G>A, also as IVS 27+1G>A) that converted the 5' end of intron 27 from GT to AT. This mutation was found in both 2 affected individuals but 9 unaffected relatives and the 50 controls were not observed, which was consistent with the clinical diagnosis. This mutation (c.1875+1G>A) appeared to be novel, which is neither reported in literature nor registered in the Database of Collagen Mutations. The heterozygous states of patients' intron 27 were confirmed by PCR-SSP. CONCLUSION: We identify a novel RNA-splicing mutation (c.1875+1G>A) in COL1A1 gene resulting in OI type I in a Chinese family. The detailed molecular and clinical features will be useful for extending the evidence for genetic and phenotypic heterogeneity in OI and exploring the phenotype-genotype correlations in OI.

[Direct multiplex-PCR from whole blood for rapid detection of Y chromosome microdeletions].

OBJECTIVE: To establish a rapid and simple method to detect Y chromosome microdeletions directly using whole blood as starting material for multiplex-PCR. METHODS: Using a self-prepared pHpH-Bufferq, multiplex-PCR amplification was directly carried out from the anticoagulant whole blood sample without DNA extraction step. Twelve sequence tagged sites (STS), namely SY84, SY86, SY127, SY134, SY124, SY132, SY152, SY157, SY239, SY242, SY254 and SY255, in AZFa, AZFb, and AZFc gene regions were detected in 5 different tubes. In order to ensure the validity of the experiments, sex-determining region Y (SRY) and X-linked or Y-linked zinc finger gene (ZFX/Y) were used as internal controls. Furthermore, conventional PCR using genomic DNA extracted from each blood sample was performed in parallel for evaluating the accuracy of the experiments. RESULTS: A total of 156 male samples were detected, and a normal male sample and a female sample were used as a positive and a negative control respectively. The results showed that 144 samples had no deletion; one sample was AZF-deleted; one sample was AZFb-deleted; seven samples were AZFc-deleted; one sample was both AZFb- and AZFc- deleted; and two samples were all AZFa-, AZFb- and AZFc- deleted. The observed results from two kinds of starting material (whole blood and purified DNA) are completely consistent. CONCLUSION: In our method, PCR amplification was directly carried out from whole blood without any DNA extraction step. So it has the advantages of low cost, simple process, time-saving operation and less cross-contamination. The whole process can be completed within 2 hours. Thus the efficiency of clinical detection is improved greatly.

[AZF microdeletions are not related with recurrent spontaneous abortion].

OBJECTIVE: To study the relationship between azoospermia factor (AZF) microdeletions of the Y-chromosome and recurrent spontaneous abortion. METHODS: We collected 26 chorionic villus samples from abortive male embryos and 51 blood samples from the husbands whose wives had recurrent spontaneous abortion, extracted genomic DNA from the samples and detected 12 STSs in the AZFa, AZFb and AZFc regions of Yq11.2 by amplification multiplex PCR. RESULT: AZF microdeletions were found neither in the chorionic villus samples nor in the blood samples. CONCLUSION: There is no relationship between AZF microdeletions and recurrent spontaneous abortion.

[Evaluation of CD4+ CD25+ regulatory T cells in the peripheral blood of recurrent spontaneous abortion patients].

OBJECTIVE: To explore the role of CD4+ CD25+ regulatory T cells (CD4+ CD25+ Tr) in the pathogenesis of recurrent spontaneous abortion. METHODS: Peripheral blood samples were collected from 29 women with unexplainable recurrent spontaneous abortion (the URSA group) and another 20 with normal pregnancy (the control group). The percentage of CD4+ CD25+ Tr in the peripheral blood was measured by flow cytometry. RESULTS: The rate of CD4+ CD25bright Tr in the URSA patients ([1.98 +/- 0.96]%) was significantly lower than that in the control group ([3.21 +/- 1.25]%, P < 0.05), while the percentages of CD4+ CD25+ and CD4+ CD25dim and the ratio of CD4+ CD25bright/CD4+ were not significantly different between the two groups (P > 0.05). CONCLUSION: URSA might be associated with the decreased percentage of CD4+ CD25bright Tr, which plays an important role in fetomaternal immunologic tolerance.

[Prenatal diagnosis of two pregnancies with risk of chromosomal disorders].

OBJECTIVE: To report the prenatal diagnosis of 2 cases of pregnancy with the risk of chromosomal disorders. In Case 1, the pregnant woman had a daughter with testicular regression syndrome and a segmental duplication of Ypter --> Yp11.2 and a deletion of Yq11.23 --> Yqter. In Case 2, both the pregnant woman and her husband were carriers of chromosomal balanced translocation. METHODS: Two samples of amniotic fluid were obtained at the 19th week of gestation for fetal karyotype analysis. For Case 1, FISH with a probe of Xp/Yp subtelomere was performed on the metaphase of the amniotic fluid, genomic DNA of the amniotic fluid extracted and multiplex PCR conducted for AZF regions. Both the pregnant women underwent sonography to confirm the karyotypic diagnosis. RESULTS: Cytogenetic, FISH and multiplex PCR analysis of the cultured amniotic fluid cells from Case 1 showed a normal male karyotype, and ultrasound scan of the fetus showed normal male external genitalia and normal development. Cytogenetic analysis of the cultured amniotic fluid cells from Case 2 revealed a karyotype of balanced translocation with t(13 ; 14) from the father, and no abnormality of the fetus was found by ultrasound scan. CONCLUSION: It is helpful to perform cytogenetical and molecular prenatal diagnosis in combination with ultrasound scan for the fetus with the risk of chromosomal disorders and subsequently for genetic counseling.

[Clinical, molecular and cytogenetic studies on 4 patients with 46, XX (SRY positive) male syndrome].

OBJECTIVE: To analyze the clinical, molecular and cytogenetic features of 46, XX (SRY positive) male syndrome. METHODS: The clinical features of 4 patients with 46, XX (SRY positive) male syndrome were analyzed retrospectively. Karyotyping, FISH, PCR amplification of the SRY gene, and Y-chromosome microdeletion were performed to study their molecular cytogenetic features. RESULTS: The Four patients were all sociopsychologically males of short stature and came to hospital for infertility. Physical examination revealed that their testes were small in volume and soft in texture, but their penes were normal. Semen analyses showed complete azoospermia. Detection of serum sexual hormone suggested hypergonadotropic hypogonadism. All were karyotyped as 46, XX. Molecular analyses revealed the presence of the SRY gene and absence of AZFa, b and c of the Y chromosome. FISH analysis showed that SRY genes were translocated to Xp in 3 of the patients. CONCLUSION: Phenotypically 46, XX (SRY positive) male patients are males generally, for the presence of the SRY gene in the whole genome and azoospermia due to the deletion of AZF. The clinical characteristics of the patient include testis dysgenesis, infertility and short stature. The long arm of the Y chromosome might contain the gene associated with body height. Extensive molecular and cytogenetic studies on 46, XX male syndrome may help to elucidate its genotype-phenotype relation.