Identification of novel TMEM231 gene splice variants and pathological findings in a fetus with Meckel Syndrome.

Background: Meckel Syndrome (MKS, OMIM #249000) is a rare and fatal autosomal recessive ciliopathy with high clinical and genetic heterogeneity. MKS shows complex allelism with other related ciliopathies such as Joubert Syndrome (JBTS, OMIM #213300). In MKS, the formation and function of the primary cilium is defective, resulting in a multisystem disorder including occipital encephalocele, polycystic kidneys, postaxial polydactyly, liver fibrosis, central nervous system malformations and genital anomalies. This study aimed to analyze the genotype of MKS patients and investigate the correlation between genotype and phenotype. Methods: A nonconsanguineous couple who conceived four times with a fetus affected by multiorgan dysfunction and intrauterine fetal death was studied. Whole exome sequencing (WES) was performed in the proband to identify the potentially pathogenic variant. Sanger sequencing was performed in family members. In silico tools were used to analyse the pathogenicity of the identified variants. cDNA TA-cloning sequencing was performed to validate the effects of intronic variants on mRNA splicing. Quantitative real-time PCR was performed to investigate the effect of the variants on gene expression. Immunofluorescence was performed to observe pathological changes of the primary cilium in kidney tissue from the proband. Results: Two splice site variants of TMEM231 (NM_001077418.2, c.583-1G>C and c.583-2_588delinsTCCTCCC) were identified in the proband, and the two variants have not been previously reported. The parents were confirmed as carriers. The two variants were predicted to be pathogenic by in silico tools and were classified as pathogenic/likely pathogenic variants according to the American College of Medical Genetics and Genomics guideline. cDNA TA cloning analysis showed that both splice site variants caused a deletion of exon 5. RT-PCR revealed that the expression of TMEM231 was significantly decreased and immunofluorescence showed that the primary cilium was almost absent in the proband's kidney tissue. Conclusion: We reported the clinical, genetic, molecular and histochemical characterisation of a family affected by MKS. Our findings not only extended the mutation spectrum of the TMEM231 gene, but also revealed for the first time the pathological aetiology of primary cilia in humans and provide a basis for genetic counselling of the parents to their offspring.

[Expert consensus on the genetic diagnosis for Dystrophinopathies].

Dystrophinopathies, including Duchenne muscular dystrophy, Becker muscular dystrophy and dilated cardiomyopathy, are X-linked recessive genetic disorders due to variants of the dystrophin gene, which can seriously affect quality of life and health. Genetic diagnosis plays a crucial role in their diagnosis, treatment, and prevention. How to rationally select and standardize the use of various genetic techniques is a skill that clinicians must acquire. By compiling expertise of experts from the relevant areas and guidelines published home and abroad, this consensus has provided a guidance from the perspective of genetic diagnosis for the selection of genetic techniques, testing strategies, and detection process for dystrophinopathies.

[Genetic analysis of a Chinese pedigree with Cohen syndrome due to compound heterozygous variants of VPS13B gene].

OBJECTIVE: To investigate the clinical phenotype and genetic characteristics of a Chinese pedigree affected with Cohen syndrome. METHODS: A proband who was admitted to Zhengzhou People's Hospital on June 2, 2021 due to intellectual disability and developmental delay, in addition with her younger sister and other family members, were selected as the study subjects. Clinical data of the proband and her younger sister were collected. Genomic DNA was extracted from peripheral venous blood and chorionic villi samples. Chromosomal abnormalities were detected with chromosomal microarray analysis (CMA). Whole exome sequencing (WES) and Sanger sequencing were carried out to detect candidate variants in the proband. With RNA extracted from the peripheral blood samples, VPS13B gene transcripts and expression were analyzed by PCR and real-time quantitative PCR. Prenatal diagnosis was carried out at 12 weeks' gestation. RESULTS: The proband was a 10-year-old female with clinical manifestations including development delay, obesity, severe myopia and peculiar facial features. Her sister was 3 years old with a similar phenotype. CMA revealed no chromosomal abnormality in the proband, while WES results revealed that the proband and her sister had both harbored compound heterozygous variants of the VPS13B gene, namely c.10076_10077delCA (p.T3359fs*29) and c.6940+1G>T, which were respectively inherited from their mother and father. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were classified as pathogenic (PVS1+PS4+PM4+PP1; PVS1+PM2_Supporting+PM3+PP1). In vivo splicing assay confirmed that the c.6940+1G>T variant has produced a frameshift transcript with skipping of exon 38. Compared with the control group, the expression of RNA in the peripheral blood of the proband's parents has decreased to 65% ~ 70% (P < 0.01), whilst that in the proband and her sister has decreased to 40% (P < 0.001). Prenatal diagnosis at 12 weeks of gestation has found that the fetus only harbored the heterozygous c.10076_ 10077delCA variant. CONCLUSION: The c.10076_10077delCA (p.T3359fs*29) frameshift variant and c.6940+1G>T splicing variant probably underlay the Cohen syndrome in this pedigree. Genetic testing has facilitated the diagnosis of this disease.

[Expert consensus on the application of prenatal exome sequencing for fetal structural anomalies].

Prospective research have shown that whole exome sequencing (WES) may be considered when a diagnosis cannot be obtained using routine prenatal methods, e.g., chromosomal karyotyping and copy number variation sequencing, for fetuses with significant structural anomalies. WES can increase the diagnostic rate of genetic disorders in such fetuses by 8% - 10%. Prenatal WES has been gaining wide acceptance. However, due to the limitations of fetal phenotypic evaluation and complexity of ethical issues in prenatal diagnosis, to justify and standardize the application of prenatal WES and maximize its clinical utility has become an urgent need. In view of this, a consensus has been formed by referring to the latest guidelines, expert consensus and authoritative literature. This consensus has put forward suggestions on the suitable objects of prenatal WES, pre-test consultation, sampling and laboratory testing, results report, post-test consultation, pregnancy outcome follow-up, multidisciplinary consultation of difficult cases, preservation of prenatal WES samples and data information.

[Analysis of CNTNAP1 gene variants in a Chinese pedigree affected with lethal congenital contracture syndrome type 7].

OBJECTIVE: To explore the genetic basis for a couple who had developed polyhydramnios during three pregnancies and given birth to two liveborns featuring limb contracture, dyspnea and neonatal death. METHODS: Whole-exome sequencing (WES) was carried out on fetal tissue and peripheral blood samples from the couple. Suspected variants were verified by Sanger sequencing. RESULTS: The fetus was found to harbor homozygous nonsense c.3718C>T (p.Arg1240Ter) variants of the CNTNAP1 gene, which were respectively inherited from its mother and father. The variant was unreported previously. According to the guidelines of the American College of Medical Genetics and Genomics, the variant was predicted to be pathogenic (PVS1+PM2+PP4). CONCLUSION: The novel homozygous nonsense variants of the CNTNAP1 gene probably underlay the lethal congenital contracture syndrome type 7 (LCCS7) in this pedigree. Above finding has enabled genetic counseling and prenatal diagnosis for the family.

[Analysis of a Chinese pedigree affected with dyschromatosis symmetrica hereditaria due to a novel variant of ADAR gene].

OBJECTIVE: To explore the genetic basis for a Chinese pedigree affected with dyschromatosis symmetrica hereditaria (DSH). METHODS: PCR and Sanger sequencing were carried out for the proband, and suspected variant was validated by Sanger sequencing in the pedigree. RESULTS: The proband was found to harbor a novel variant of c.1352delA (p.N451Mfs*13) of the ADAR (NM_001111) gene. The same variant was found in her affected mother and sister, but not in her unaffected father, uncle, and 100 healthy individual. CONCLUSION: The novel variant of the ADAR gene probably underlay the pathogenesis of DSH in this pedigree.

[Identification of SPAST gene variant in a pedigree affected with hereditary spastic paraplegia type 4].

OBJECTIVE: To explore the genetic basis for a pedigree affected with hereditary spastic paraplegia type 4 (HSP4). METHODS: Peripheral venous blood samples were taken from members of the four-generation pedigree and 50 healthy controls for the extraction of genomic DNA. Genes associated with peripheral neuropathy and hereditary spastic paraplegia were captured and subjected to targeted capture and next-generation sequencing. The results were confirmed by Sanger sequencing. RESULTS: DNA sequencing suggested that the proband has carried a heterozygous c.1196C>G variant in exon 9 of the SPAST gene, which can cause substitution of serine by threonine at position 399 (p.Ser399Trp) and lead to change in the protein function. The same variant was also detected in other patients from the pedigree but not among unaffected individuals or the 50 healthy controls. Based on the ACMG 2015 guidelines, the variant was predicted to be possibly pathogenic. CONCLUSION: The c.1196C>G variant of the SPAST gene probably underlay the HSP4 in this pedigree.

[Prenatal diagnosis of a fetus affected with Finnish type congenital nephrotic syndrome].

OBJECTIVE: To explore the genetic basis for a fetus suspected for congenital nephrotic syndrome of Finland (CNF). METHODS: Genomic DNA was extracted from peripheral and umbilical cord blood samples derived from both parents and the fetus. Potential variants were detected by using next-generation sequencing. Suspected variants were confirmed by Sanger sequencing. RESULTS: The fetus was found to carry compound heterozygous variants c.1440+1G>A and c.925G>T of the NPHS1 gene, which were respectively inherited from its mother and father. CONCLUSION: Identification of the compound heterozygous NPHS1 variants has enabled diagnosis of CNF in the fetus and genetic counseling for the affected family.

[Genetic diagnosis of a child with Smith-Magenis syndrome].

OBJECTIVE: To explore the molecular mechanism of a girl with developmental delay and intellectual disability. METHODS: Chromosomal karotypes of the child and her parents were analyzed with routine G-banding method. Their genomic DNA was also analyzed with array comparative genomic hybridization (aCGH) for chromosomal duplications/deletions. RESULTS: No karyotypic abnormality was detected in the child and her parents, while aCGH has identified a de novo 3.37 Mb deletion at 17p11.2 in the child. CONCLUSION: The child was diagnosed with Smith-Magenis syndrome, for which RAI1 may be the causative gene.

[Recurrent Angelman syndrome caused by a rare partial deletion of UBE3A gene].

OBJECTIVE: To provide genetic testing for two brothers with mental retardation and epilepsy. METHODS: Array comparative genomic hybridization (aCGH) was used to detect copy number variations in the two patients, their parents and maternal grandparents. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was utilized to delineate the deleted region in the pedigree. RESULTS: A 138 kb deletion in 15q11.2 region was detected by aCGH in both patients, which encompassed part of the UBE3A gene. MS-MLPA has narrowed down the region to exons 8 to 14 of the UBE3A gene. The same deletion was also found in their mother and grandfather. CONCLUSION: The pathogenesis of this rare form of recurrent Angelman syndrome may be attributed to the partial deletion of maternal UBE3A gene.

[Prenatal diagnosis and genetic analysis of a fetus with 2p13.3-p12 microdeletion].

OBJECTIVE: To provide prenatal diagnosis for a pregnant woman with a history of Williams-Beuren syndrome pregnancy. METHODS: The karyotypes of the fetus and his parents were analyzed with routine G-banding. Their genomic DNA was also analyzed with array comparative genomic hybridization (aCGH). RESULTS: No karyotypic abnormality was detected for the fetus and his parents. aCGH has identified a de novo 5.09 Mb deletion at 2p13.3-p12 in the fetus. CONCLUSION: The 2p13.3-p12 microdeletion carried by the fetus was de novo. As it has involved dosage-sensitive genes SPR and DCTN1, the deletion is probably pathogenic.

Targeted next-generation sequencing-based molecular diagnosis of congenital hand malformations in Chinese population.

Congenital hand malformations is rare and characterized by hand deformities. It is highly heterogeneous, both clinically and genetically, which complicates the identification of causative genes and mutations. Recently, targeted next-generation (NGS) sequencing has been successfully used for the detection of heterogeneous diseases, and the use of NGS also has contributed significantly in evaluating the etiology of heterogeneous disease. Here, we employed targeted NGS to screen 248 genes involved in genetic skeletal disorders, including congenital hand malformations. Three pathogenic mutations located in the GJA1, ROR2 and TBX5 genes were detected in three large Chinese families with congenital hand malformations. Two novel mutations were reported, and a known causative mutation was verified in this Chinese population. This is also the first report that the same panel of targeted NGS was employed to perform molecular diagnosis of different subtypes of congenital hand malformations. Our study supported the application of a targeted NGS panel as an effective tool to detect the genetic cause for heterogeneous diseases in clinical diagnosis.

[Genetic diagnosis of a child with aortic stenosis and thumb aplasia].

OBJECTIVE: To analyze the molecular mechanism and prognosis of a child with aortic stenosis and thumb aplasia. METHODS: The karotypes of the child and his parents were analyzed with routine G-banding. Their genomic DNA was also analyzed with array comparative genomic hybridization(aCGH) for chromosomal duplications/deletions. RESULTS: No karyotypic abnormality was detected at cytogenetic level for the child and his parents. aCGH identified a de novo 5.86 Mb deletion at 2q22.3-q23.3 in the child. CONCLUSION: The child was diagnosed with 2q23.1 microdeletion syndrome. MBD5 may be the key gene for the 2q23.1 microdeletion syndrome.

[Prenatal diagnosis and genetic analysis of a fetus with endocardial cushion defects].

OBJECTIVE: To perform prenatal diagnosis for a fetus with endocardial cushion defect and explore its mechanism. METHODS: The karotypes of the fetus and its parents were analyzed by routine G-banding. Their genomic DNA was also analyzed by array comparative genomic hybridization (aCGH). RESULTS: The fetus and its mother were found to have a karyotype of 46, XX, inv(8)(p21q24.1), while no karyotypic abnormality was detected for the father. aCGH has detected a 15.14 Mb deletion at 8p23.3-p22 and a 6.87 Mb duplication at 8q24.23-q24.3 in the fetus. CONCLUSION: The fetus was diagnosed with Rec8 syndrome. Its abnormal chromosomes have derived from the inv(8) carried by its mother. GATA4 and SOX7 may be the key genes for the endocardial cushion defect found in the fetus.

[Analysis of WAS gene mutation in a Chinese family affected with Wiskott-Aldrich syndrome].

OBJECTIVE: To detect potential mutation of the WAS gene in a Chinese family affected with Wiskott-Aldrich syndrome. METHODS: Peripheral blood samples were collected from the proband and his family members. All exons and flanking regions of the WAS gene were subjected to PCR amplification - Sanger sequencing as well as restriction endonuclease analysis. Plasma level of B-cell activating factor (BAFF) was also determined for all family members. RESULTS: A hemizygous mutation (c.257G>A) of the WAS gene was identified in all patients from the family, for which the patient's mother was heterozygous. The same mutation was not found among healthy members of the family. Compared with unaffected members, all patients had a higher level of BAFF. CONCLUSION: The c.257G>A mutation of the WAS gene probably underlies the Wiskott-Aldrich syndrome in this family.

[Genetic analysis of a child with cleidocranial dysplasia and 6q21-q22.31 microdeletion].

OBJECTIVE: To carry out genetic analysis on a child with developmental delay and multiple malformation. METHODS: The karotypes of the child and her parents were analyzed with routine chromosomal G-banding. Their genomic DNA was analyzed with array comparative genomic hybridization (aCGH). RESULTS: The karyotype of the proband was determined as 46,XX,del(6)(q22),inv(6)(p21.1q21), while no karyotypic abnormality was detected in her parents. aCGH has identified in the child a de novo 800 kb deletion encompassing the RUNX2 gene at 6p21.1 and a de novo 11.79 Mb deletion at 6q21-q22.31. CONCLUSION: Both of the de novo deletions are pathogenic. Deletion of the RUNX2 gene probably underlies the cleidocranial dysplasia in the patient, while the 6q21-q22.31 deletion may result in malformation of the brain.

[Identification of a novel EXT1 mutation in a pedigree affected with hereditary multiple exostosis].

OBJECTIVE To detect potential mutations of the EXT1 and EXT2 genes in a pedigree affected with hereditary multiple exostosis (HME). METHODS For a four-generation family with 7 affected individuals from 17 family members,genomic DNA was extracted from peripheral venous blood samples. All exons of the EXT1 and EXT2 genes were screened for potential mutation by PCR and Sanger sequencing. RESULTS A novel heterozygous frameshift mutation c.1202delT (p.I401Tfs*2)was found in exon 4 of the EXT1 gene in the proband and the other 6 affected individuals. The same mutation was not detected among the healthy members from the family. The mutation has given rise a truncated EXT1 protein with loss of 345 amino acids. CONCLUSION A novel frameshift mutation of the EXT1 gene has been identified in a pedigree affected with HME, which has enriched the mutational spectrum of the EXT1 gene and may facilitate genetic counseling and prenatal diagnosis for the family.

[Clinical and genetic analysis of a boy with 9q34.3 microdeletion syndrome].

OBJECTIVE: To determine the origin of chromosomal aberration in a boy with mental retardation and multiple congenital malformations. METHODS: The karotypes of the proband and his parents were analyzed with conventional G-banding. Their genomic DNA was analyzed with array comparative genomic hybridization (aCGH). RESULTS: No karyotypic abnormality was detected in the proband and his parents. aCGH has identified a de novo 405 kb deletion at 9q34.3 in the proband, which encompassed the EHMT1 gene and part of CACNA1B gene. CONCLUSION: The de novo 9q34.3 deletion probably underlies the mental retardation and development delay in the boy. EHMT1 may be one of the key genes responsible for 9q34.3 microdeletion syndrome.

[Prenatal diagnosis and genetic analysis of a fetus with 6q27 microdeletion].

OBJECTIVE: To determine the origin and pathogenicity of a chromosomal aberration for a fetus and analyze the possible mechanism. METHODS: The karotypes of the fetus and its parents were analyzed with routine G-banding. Their genomic DNA was also analyzed with array comparative genomic hybridization (aCGH). RESULTS: No karyotypic abnormality was detected at cytogenetic level for the fetus and its parents. aCGH has identified a de novo 2.04 Mb deletion at 6q27 in the fetus. The region involves candidate genes responsible for structural brain abnormalities. The area flanking the chromosomal breakpoint contains a 2410 bp sequence rich in palindromes which can form stable secondary structures. CONCLUSION: The de novo 6q27 deletion is pathogenic. The 6q27 deletion may be responsible for the structural brain abnormalities in the fetus. The palindrome sequence flanking the chromosomal breakpoint may be involved the formation of the 6q27 deletion.

Genotype/phenotype analysis in a male patient with partial trisomy 4p and monosomy 20q due to maternal reciprocal translocation (4;20): A case report.

Translocations are the most frequent structural aberration in the human genome. Carriers of balanced chromosome rearrangement exhibit an increased risk of abortion and/or a chromosomally­unbalanced child. The present study reported a clinical and cytogenetic analysis of a child who exhibited typical trisomy 4p and monosomy 20q features, including intellectual disability, delayed speech, tall stature, seizures and facial dysmorphism. The karyotype of the proband exhibited 46, XY, add(20) (q13.3). The karyotype of the mother indicated a balanced translocation karyotype: 46, XX, t(4;20) (p15.2;q13.1). The array­based comparative genomic hybridization (aCGH) analysis identified partial trisomy of the short arm of chromosome 4 and partial monosomy of distal 20q in the proband due to maternal balanced reciprocal translocation 4;20. The analysis of genotype/phenotype correlation demonstrated that fibroblast growth factor receptor 3 and msh homeobox 1 may be the important genes for 4p duplication, and that potassium voltage­gated channel subfamily Q member 2, myelin transcription factor 1 and cholinergic receptor nicotinic α4 subunit may be the important genes for 20q deletion. To the best of our knowledge, the present study was the first to report an unbalanced translocation involving chromosomes 4p and 20q. The present study additionally demonstrated that aCGH analysis is able to reliably detect unbalanced submicroscopic chromosomal aberrations.