[Genetic analysis of a fetus with Meckel syndrome due to variants of TMEM67 gene].

OBJECTIVE: To carry out prenatal diagnosis for a fetus with Meckel syndrome (MKS) and explore its genetic basis. METHODS: A pregnant woman presented at Suzhou Municipal Hospital in February 2018 was selected as the study subject. Clinical data was collected. Muscle tissue sample from the abortus and peripheral blood samples from the couple were collected. Genomic DNA was extracted and subjected to chromosomal microarray analysis (CMA) and whole exome sequencing. Candidate variant was verified by Sanger sequencing. RESULTS: The fetus was found to have microcephaly, oligohydramnios, polycystic kidneys and banana-shaped cerebellum at 18 weeks of gestation. After induction of labor, it was found to have encephalocele, renal cysts and polydactyly. CMA has found no abnormality. Whole exome sequencing revealed novel compound heterozygous variants c.296delA (p.Lys99SerfsTer6) and c.1243G>A (p.Val415Met) in the TMEM67 gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.296delA variant was predicted to be pathogenic (PVS1+PM2_Supporting+PP4), whilst the c.1243G>A variant was predicted to be likely pathogenic (PM2_Supporting+PM3+PP3_Moderate+PP4). CONCLUSION: The c.296delA and c.1243G>A compound heterozygous variants of the TMEM67 gene probably underlay the MKS in this fetus.

Novel homozygous mutations in TXNDC15 causing Meckel syndrome.

BACKGROUND: Meckel syndrome (MKS) is the most severe form of an autosomal recessive ciliopathy and is clinically characterized by occipital encephalocele, severely polycystic kidneys, and postaxial polydactyly (toes). The association of TXNDC15-related MKS has been reported. We report the case of a homozygous mutation in the TXNDC15 gene, causing MKS14 in the Chinese population. METHODS: The fetal skin tissue and parental peripheral blood were retained for whole-exome sequencing and Sanger sequencing, which investigated the potential pathogenic variants associated with MKS. RESULTS: The fetus was homozygous for a mutation in the TXNDC15 gene (NM_024715.3), specifically c.560delA (p.Asn187llefsTer4), and both parents were heterozygous for this mutation. CONCLUSION: Our study identified a new mutation that adds to the mutational landscape of MKS, which provide a basis for genetic counseling and the selection of reproductive options.

Homozygous LOXL2 variant in individuals affected by non-syndromic occipital encephalocele.

BACKGROUND: Occipital encephaloceles is a rare congenital defect in which meninges and the brain protrude out as a sac-like structure through opening in the skull. The condition can result in neurologic complications as well as structural abnormalities of the skull. To the best of our knowledge, no genetic variant has been identified as an underlying cause of non-syndromic occipital encephaloceles. METHODS: In this study, I report a family with 2 individuals having large occipital encephalocele. Clinical and radiological examination did not reveal any other neurological or skeletal manifestations in both affected individuals. RESULTS: Exome sequencing detected the previously unreported homozygous single nucleotide duplication (NM_002318.3:c.64dupC) in LOXL2 gene. This is a nonsense variant (NP_002309.1:p.Leu22Profs*7) leading to a premature truncation and loss-of-function of the lysyl oxidase-like 2 protein. The variant is segregating in an autosomal recessive manner in a family. Both parents are heterozygous carriers for the variant while unaffected sibs have wild type sequence. CONCLUSION: We hypothesize that LOXL2 is a potential candidate gene for occipital encephalocele due to the established role of LOXL3, a close paralog of LOXL2, in craniofacial development. This case illustrates the power of exome sequencing to establish genetic diagnosis and expands the spectrum of genetic defects in occipital encephalocele and related disorders.

A non-coding insertional mutation of Grhl2 causes gene over-expression and multiple structural anomalies including cleft palate, spina bifida and encephalocele.

Orofacial clefts, including cleft lip and palate (CL/P) and neural tube defects (NTDs) are among the most common congenital anomalies, but knowledge of the genetic basis of these conditions remains incomplete. The extent to which genetic risk factors are shared between CL/P, NTDs and related anomalies is also unclear. While identification of causative genes has largely focused on coding and loss of function mutations, it is hypothesized that regulatory mutations account for a portion of the unidentified heritability. We found that excess expression of Grainyhead-like 2 (Grhl2) causes not only spinal NTDs in Axial defects (Axd) mice but also multiple additional defects affecting the cranial region. These include orofacial clefts comprising midline cleft lip and palate and abnormalities of the craniofacial bones and frontal and/or basal encephalocele, in which brain tissue herniates through the cranium or into the nasal cavity. To investigate the causative mutation in the Grhl2Axd strain, whole genome sequencing identified an approximately 4 kb LTR retrotransposon insertion that disrupts the non-coding regulatory region, lying approximately 300 base pairs upstream of the 5' UTR. This insertion also lies within a predicted long non-coding RNA, oriented on the reverse strand, which like Grhl2 is over-expressed in Axd (Grhl2Axd) homozygous mutant embryos. Initial analysis of the GRHL2 upstream region in individuals with NTDs or cleft palate revealed rare or novel variants in a small number of cases. We hypothesize that mutations affecting the regulation of GRHL2 may contribute to craniofacial anomalies and NTDs in humans.

NID1-related autosomal dominant Dandy-Walker malformation with occipital cephalocele in three generations.

The combination of Dandy-Walker malformation and occipital cephalocele is a rare autosomal dominant condition, known as ADDWOC, and caused by mutations in NID1 or LAMC1. We present a three-generation family with variable manifestations of Dandy-Walker malformation and occipital cephalocele. They all have normal psychomotor development and lack neurological manifestations. Mutation analysis revealed a likely pathogenic missense variant in NID1 (c.3336T > G, p.Asn1112Lys), affecting an amino acid residue crucial in the nidogen/laminin interaction.

ERF-related craniosynostosis and surgical management in the paediatric cohort.

INTRODUCTION: ERF mutation is one of the most recently identified genetic aberrations associated with syndromic craniosynostosis. Data on the pattern of craniosynostosis, surgical management of ERF-related craniosynostosis and outcomes is limited. We report on our single-centre experience in paediatric cohort of patients with syndromic craniosynostosis secondary to ERF mutation. METHODS: A retrospective review of all paediatric craniofacial cases was performed over an 8-year period (2014-2022). All patients with genetically confirm ERF-related craniosynostosis were identified, and clinical parameters including, age, sex, pattern of craniosynostosis, associated tonsillar herniation and follow-up period were further analysed from electronic clinical and imaging systems. All patients were selected and discussed in multidisciplinary craniofacial meeting (composed of neurosurgical, maxillofacial, plastics and genetics teams) prior to any surgical intervention. RESULTS: Overall, 10 patients with ERF-related craniosynostosis were identified with a male-to-female ratio of 4:1 with mean age at the time of surgery of 21.6 months with a mean follow-up period of 5.2 years. ERF-confirmed cases led to variable craniosynostosis pattern with multi-sutural synostosis with concurrent sagittal and bilateral lambdoid involvement as the most common pattern (7/10). No patient pre-operatively had evidence of papilloedema on ophthalmological assessment. Eight out of 10 patients had associated low-lying tonsils/hind brain hernia pre-operatively. Eight out of 10 patients required surgery which included 2 fronto-orbital advancement, 3 calvarial remodelling, 2 posterior calvarial remodelling/release and 1 insertion of ventriculoperitoneal shunt. CONCLUSION: Involvement of sagittal and lambdoid sutures is the most common pattern of craniosynostosis. ERF-related craniosynostosis can have variable pattern of suture fusion, and management of each patient requires unique surgical planning and execution based on clinical needs for the optimal outcomes.

[Knobloch syndrome: a case report].

A 5-year-old girl came to the Tianjin Medical University Eye Hospital in May 2021 because of her poor eyesight after birth. The physical examination showed that she had high myopia, esotropia, horizontal tremor, and high myopia retinopathy of both eyes. After inquiring about her medical history, we found that the baby's occipital cystic mass swelled after birth, and CT examination showed that the occipital skull plate defect with meningocele, but without treatment, at present, the occipital mass had subsided by itself. Considering the eye manifestations and skull changes of the child, it may be conformed to Knobloch syndrome, after the detection of V4 by full exon gene, it was found that the child had the compound heterozygous variation of pathogenic gene COL18A1, and Knobloch syndrome was definite, Knobloch syndrome is a rare autosomal recessive hereditary disease with typical features of high myopia, retinal detachment and occipital encephalocele. At present, there is no clear treatment plan, and gene therapy may be an effective treatment for Knobloch syndrome in the future.

Evaluation of novel compound variants of CEP290 in prenatally suspected case of Meckel syndrome through whole exome sequencing.

BACKGROUND: Meckel syndrome (MKS) is a fatal disease characterized by multisystem fibrosis during the prenatal or perinatal period. It has an autosomal recessive genetic pattern and is characterized by meningo occipital encephalocele, polycystic kidney dysplasia, polydactyly, and hepatobiliary ductal plate malformation. Germline variations in CEP290 have been shown to cause MKS4. METHODS: In this study, a 23-year-old Chinese woman who was 18 weeks pregnant was examined. The pregnancy was terminated due to occipital meningocele and enlarged cystic dysplastic kidney revealed by ultrasonography. In addition, the patient had a history of adverse pregnancy whereby the fetus presented with double kidney enlargement. Karyotype analysis and chromosomal microarray examination (CMA) were carried out using amniotic fluid samples. Whole exome sequencing (WES) was performed using tissue specimens of the aborted fetus. RESULTS: Karyotype and CMA analyses showed normal results. However, compound heterozygous mutations of CEP290 c.3175dup and CEP290 c.1201dup were detected through WES. CEP290 c.1201dup is a novel heterozygous mutation of CEP290 that has not been reported previously. CONCLUSIONS: The findings of this study provide information on the correlation between MKS phenotype and genotype in CEP290. In addition, these findings indicate that WES is an effective method for detecting genetic causes of multiple structural defects especially those showing normal karyotype and CMA results.

Identification of Pathogenic Variants in RPGRIP1L with Meckel Syndrome and Preimplantation Genetic Testing in a Chinese Family.

Meckel syndrome (MKS, OMIM:249000) is a severe multiorgan dysplastic lethal ciliopathy with extreme genetic heterogeneity. Defects in RPGRIP1L are the cause of MKS type 5 (MKS5, OMIM:611561). However, only six different variants have been reported in eight MKS5 cases with biallelic variants. Here, we describe the case of a Chinese family with recurrent fetal malformations. The proband was a 14-week gestation fetus with occipital encephalocele, polycystic kidneys, polydactyly, and single ventricular heart. Trio whole-exome sequencing was performed, and two novel compound heterozygous variants of RPGRIP1L (c.427C > T, p.Gln143Ter and c.1351-11A > G) were identified. cDNA studies of the splicing variant demonstrated a reading-frame shift with a subsequent premature stop codon (p.Glu451Serfs*6). After the proband was diagnosed with MKS5, the couple chose preimplantation genetic testing for monogenic disorders (PGT-M) and prenatal genetic diagnosis (PND) to prevent the transmission of pathogenic variants, which led to a successful pregnancy recently. In summary, we have identified two novel variants of RPGRIP1L in a Chinese family, which expand the variant spectrum of MKS5. Furthermore, we have described the successful application of PGT-M and PND in this family. These techniques could assist couples with a genetic predisposition in avoiding the transmission of genetic diseases to their offspring.

Prenatally detected encephalocele associated with a novel pathogenic TCTN3 variant: A case report and literature review.

Primary cilia are a component of almost all vertebrate cells with a crucial role in sensing and transducing environmental signals during tissue development. Their dysfunction is known as ciliopathies and can manifest with a wide spectrum of clinical disorders. Overlapping features and molecular heterogeneity of ciliopathies make diagnoses distinctly challenging. In this group of diseases, tectonic genes, and their mutations play an important role. We present a first-trimester fetus with occipital encephalocele and OFD type IV caused by TCTN3 compound heterozygous pathogenic variants: c.1423_1429del (p.Arg475Serfs*10) and c.3G>A (initiator codon). A severe arm anomaly was described in our case, with two fingers along the atrophic forearm and polydactyly on other limbs. This could be a new phenotypic characteristic contributing to further understanding of TCTN3-related disorders as well as other tectonic proteins in ciliopathy spectrum diseases.

Three Novel Variants of CEP290 and CC2D2DA and a Link Between ZNF77 and SHH Signaling Pathway Are Found in Two Meckel-Gruber Syndrome Fetuses.

Meckel-Gruber syndrome (MKS) is a rare lethal autosomal recessive inherited disorder. Missed diagnosis might happen in clinical works due to an unclear genotype-phenotype correlation. We analyzed two families visiting our center; the parents are normal; each of the family aborted a fetus at 12WG. Following ultrasonography and pathological examination, both were diagnosed as MKS. Whole exome sequencing identified a compound heterozygous of two novel variants of CEP290 and a heterozygous of a novel variant of CC2D2A. Frameshift mutations in ZNF77 were also detected. Western blot analyzing whole-brain tissue showed that the expression of ZNF77, CC2D2A, and CEP290 was enhanced. HEK293T transfected with over-expression wildtype/mutated ZNF77 plasmid showed that SHH was increased in wildtype ZNF77 cells, while SHH and CC2D2A were increased in mutated ZNF77 cells. Our research provided two novel pathogenic variants of CEP290 and CC2D2A and suggested that ZNF77 might promote the expression of CC2D2A and regulate the amount of SHH.

HK1 haemolytic anaemia in association with a neurological phenotype and co-existing CEP290 Meckel-Gruber in a Romani family.

HK1 deficient Haemolytic Anaemia in association with a Neurological Phenotype & co-existing Meckel-Gruber due to CEP290 in a Romani family.

Meckel Gruber and Joubert Syndrome Diagnosed Prenatally: Allelism between the Two Ciliopathies, Complexities of Mutation Types and Digenic Inheritance.

Background: Antenatally detected occipital encephalocele and polycystic kidneys are a common presentation of ciliopathies like Joubert syndrome and Meckel Gruber syndrome which have considerable genetic and phenotypic overlap. Case reports: We describe 3 cases of antenatally diagnosed occipital encephalocele and enlarged kidneys with fetal autopsy, histopathology & exome sequencing results. A novel nonsense variant in the CEP290 gene was reported in first case (Meckel syndrome). The second case shows the importance of fetal exome where the parents were carriers for 2 ciliopathy genes (TMEM138 & SDCCAG8). Diagnosis in this case was confirmed by fetal exome sequencing (Joubert syndrome). Multiexon deletion in TMEM67 and KIF14 present in trans was identified in the third case (Meckel syndrome), likely resulting in digenic inheritance. Conclusion: We report 2 cases of Meckel syndrome with a novel variant and multiexon deletion, and 1 case of Joubert syndrome which depicts the limitations of preconceptional carrier screening in ciliopathies due to overlapping phenotypes.

[Genetic testing and prenatal diagnosis for a Chinese pedigree affected with Meckel-Gruber syndrome].

OBJECTIVE: To carry out genetic testing and prenatal diagnosis for a Chinese couple whom had conceived two fetuses featuring multiple malformations including polycystic kidney, polydactyly and encephalocele. METHODS: Following elective abortion, the fetus from the second pregnancy was subjected to whole exome sequencing. Suspected pathogenic variants were verified by Sanger sequencing of the fetus and its parents. RESULTS: The fetus was found to harbor compound heterozygous variants of the CEP290 gene, namely c.2743G>T (p.E915X) and c.2587-2A>T, which were respectively inherited from its father and mother. The same variants were not detected among 100 healthy controls nor reported previously. Bioinformatic analysis suggested both variants to be deleterious. The fetus was diagnosed with Meckel-Gruber syndrome. Prenatal diagnosis for the couple during their next pregnancy suggested that the fetus did not carry the above pathogenic variants. CONCLUSION: The compound heterozygous variants of the CEP290 gene probably underlay the pathogenesis of Meckel-Gruber syndrome in the second fetus. Above finding has provided a basis for genetic counseling and prenatal diagnosis for the couple, and also enriched the mutational spectrum of the CEP290 gene.

Knobloch Syndrome Associated with Novel COL18A1 Variants in Chinese Population.

Knobloch syndrome is an inherited disorder characterized by high myopia, retinal detachment, and occipital defects. Disease-causing mutations have been identified in the COL18A1 gene. This study aimed to investigate novel variants of COL18A1 in Knobloch syndrome and describe the associated phenotypes in Chinese patients. We reported six patients with Knobloch syndrome from four unrelated families in whom we identified five novel COL18A1 mutations. Clinical examination showed that all probands presented with high myopia, chorioretinal atrophy, and macular defects; one exhibited rhegmatogenous retinal detachment in one eye. Occipital defects were detected in one patient.

Prenatal Versus Postnatal Diagnosis of Meckel-Gruber and Joubert Syndrome in Patients with TMEM67 Mutations.

Renal cystic diseases are characterized by genetic and phenotypic heterogeneity. Congenital renal cysts can be classified as developmental disorders and are commonly diagnosed prenatally using ultrasonography and magnetic resonance imaging. Progress in molecular diagnostics and availability of exome sequencing procedures allows diagnosis of single-gene disorders in the prenatal period. Two patients with a prenatal diagnosis of polycystic kidney disease are presented in this article. TMEM67 mutations were identified in both fetuses using a whole-exome sequencing (WES) study. In one of them, the phenotypic syndrome diagnosed prenatally was different from that diagnosed in the postnatal period.

Meckel-Gruber Syndrome: Clinical and Molecular Genetic Profiles in Two Fetuses and Review of the Current Literature.

Background: Meckel-Gruber syndrome (MKS; OMIM No. 249000) is a rare, in utero lethal disease characterized by occipital encephalocele, polycystic kidneys, and polydactyly. Methodology and Results: In this study, two fetuses diagnosed as having MKS in the prenatal period were evaluated on the basis of ultrasonographic findings, postmortem autopsy findings, and molecular genetic analyses. Using exome sequencing analyses a novel homozygous frameshift variant (NM_015631: c.530delA, p.Lys177Argfs*47) was detected at exon 4 of TCTN3 gene in case 1, and a novel homozygous synonymous variant (NM_025114: c.180G>A, p Lys60Lys) was detected at exon 3 of CEP290 gene in case 2. Case 1 is the first reported case in the literature, which showed the typical MKS clinical feature with a novel frameshift variation in the TCTN3 gene. The variant in case 2 is the first reported synonymous variant of CEP290 gene in the literature, which has been shown to affect splicing in a functional study at the RNA level. Conclusion: TCTN3 gene variants that were rarely associated with the typical MKS phenotype and all cases with these variations have been discussed in the context of genotype-phenotype. The detection of the first synonymous variant of CEP290 gene and the demonstration of its effect on splicing by a functional study are likely to contribute to the molecular etiology of MKS.

A rare triad of morning glory disc anomaly, moyamoya vasculopathy, and transsphenoidal cephalocele: pathophysiological considerations and surgical management.

Morning glory disc anomaly is a congenital abnormality of the optic disc and peripapillary retina reported as an isolated condition or associated with various anomalies, including basal encephaloceles and moyamoya vasculopathy. However, the co-occurrence of these three entities is extremely rare and the pathogenesis is still poorly understood. Moreover, data on the surgical management and long-term follow-up of the intracranial anomalies are scarce. Here, we describe the case of a 11-year-old boy with morning glory disc anomaly, transsphenoidal cephalocele, and moyamoya vasculopathy, who underwent bilateral indirect revascularization with encephalo-duro-myo-arterio-pericranio-synangiosis at the age of 2 years, and endoscopic repair of the transsphenoidal cephalocele at the age of 6 years. A rare missense variant (c.1081T>C,p.Tyr361His) was found in OFD1, a gene responsible for a X-linked ciliopathy, the oral-facial-digital syndrome type 1 (OFD1; OMIM 311200). This case expands the complex phenotype of OFD1 syndrome and suggests a possible involvement of OFD1 gene and Shh pathway in the pathogenesis of these anomalies.

Dominant monoallelic variant in the PAK2 gene causes Knobloch syndrome type 2.

Knobloch syndrome is an autosomal recessive phenotype mainly characterized by retinal detachment and encephalocele caused by biallelic pathogenic variants in the COL18A1 gene. However, there are patients clinically diagnosed as Knobloch syndrome with unknown molecular etiology not linked to COL18A1. We studied an historical pedigree (published in 1998) designated as KNO2 (Knobloch type 2 syndrome with intellectual disability, autistic behavior, retinal degeneration, encephalocele). Whole exome sequencing of the two affected siblings and the normal parents resulted in the identification of a PAK2 non-synonymous substitution p.(Glu435Lys) as a causative variant. The variant was monoallelic and apparently de novo in both siblings indicating a likely germ-line mosaicism in one of the parents; the mosaicism, however, could not be observed after deep sequencing of blood parental DNA. PAK2 encodes a member of a small group of serine/threonine kinases; these P21-activating kinases (PAKs) are essential in signal transduction and cellular regulation (cytoskeletal dynamics, cell motility, death and survival signaling and cell cycle progression). Structural analysis of the PAK2 p.(Glu435Lys) variant that is located in the kinase domain of the protein predicts a possible compromise in the kinase activity. Functional analysis of the p.(Glu435Lys) PAK2 variant in transfected HEK293T cells results in a partial loss of the kinase activity. PAK2 has been previously suggested as an autism-related gene. Our results show that PAK2-induced phenotypic spectrum is broad and not fully understood. We conclude that the KNO2 syndrome in the studied family is dominant and caused by a deleterious variant in the PAK2 gene.