Whole exome sequencing identified a homozygous novel variant in CEP290 gene causes Meckel syndrome

Abstract Meckel syndrome (MKS) is a pre‐ or perinatal multisystemic ciliopathic lethal disorder with an autosomal recessive mode of inheritance. Meckel syndrome is usually manifested with meningo‐occipital encephalocele, polycystic kidney dysplasia, postaxial polydactyly and hepatobiliary ductal plate malformation. Germline variants in CEP290 cause MKS4. In this study, we investigated a 35‐years‐old Chinese female who was 17+1 weeks pregnant. She had a history of adverse pregnancy of having foetus with multiple malformations. We performed ultrasonography and identified the foetus with occipital meningoencephalocele and enlarged cystic dysplastic kidneys. So, she decided to terminate her pregnancy and further genetic molecular analysis was performed. We identified the aborted foetus without postaxial polydactyly. Histological examination of foetal kidney showed cysts in kidney and thinning of the renal cortex with glomerular atrophy. Whole exome sequencing identified a novel homozygous variant (c.2144T>G; p.L715*) in exon 21 of the CEP290 in the foetus. Sanger sequencing confirmed that both the parents of the foetus were carrying this variant in a heterozygous state. This variant was not identified in two elder sisters of the foetus as well as in the 100 healthy individuals. Western blot analysis showed that this variant leads to the formation of truncated CEP290 protein with the molecular weight of 84 KD compared with the wild‐type CEP290 protein of 290 KD. Hence, it is a loss‐of‐function variant. We also found that the mutant cilium appears longer in length than the wild‐type cilium. Our present study reported the first variant of CEP290 associated with MKS4 in Chinese population.


| INTRODUC TI ON
Meckel syndrome type 4 (MKS4) [MIM# 611134] is a pre-or perinatal lethal ciliopathic disorder with an autosomal recessive mode of inheritance. 1 Meckel syndrome is manifested with occipital meningoencephalocele, postaxial polydactyly, multicystic kidney dysplasia and hepatobiliary ductal plate malformation. 2 Meckel syndrome patients are usually died before or soon after birth. In addition, the incidence of MKS is 1 in 140 000 live birth worldwide. 3 In Finnish and Belgian population, the occurrence of MKS is more than the other populations. 3 Genetically, MKS is extremely heterogenous and associated with germline variants of a group of genes. 4 Moreover, MKS has been reported to be caused by the germline variants of eight genes (CEP290, MKS1, B9D1, B9D2, CC2D2A, RPGRIP1L, TMEM67, TMEM216). 4 These eight genes and their encoded proteins are playing the key role in the formation of cilia. 5 Structurally, cilia are located on the cellular surface and maintaining the structure and function of a group of cells, namely brain cells, kidney cells and liver cells. 6 Cilia are also involved in transmitting signals among adjacent cells. 6,7 So, germline variants of any of these eight genes exert effects on the structure and function of cilia which finally results into MKS.
Germline variants of CEP290 gene cause MKS4. CEP290 gene is located in the long arm (q) of chromosome 12. 8 The CEP290 gene has 54 exons and encodes CEP290 (centrosomal protein of 290 kD) protein consisting of 2479 amino acids. 8  Among those reported variants of CEP290, most of them are classified as loss-of-function (non-sense, frameshift or splice-site variants) variants. 2 In this study, we investigated a 35-years-old Chinese female who was 17+1 weeks pregnant (gravida 6, para 2). She had a history of adverse pregnancy of having foetus with multiple malformations. We performed ultrasonography and identified the foetus with all classic MKS symptoms, that is occipital meningoencephalocele, enlarged cystic dysplastic kidneys. So, she decided to terminate her pregnancy and further genetic molecular analysis was performed. We found the aborted foetus without postaxial polydactyly. Histological examination of the foetal kidney showed cysts in kidney and thinning of renal cortex with glomerular atro-

| Karyotype and chromosomal microarray analyses
In order to analyse the structure of all the chromosomes in the foetus, we performed standard G-banding karyotyping. Next, in order to confirm the presence of copy number variations (CNV) in the foetus, chromosome microarray analysis was performed using a CytoScan HD array (Affymetrix), according to the manufacturer's protocols (Affymetrix). Chromosome Analysis Suite software version 1.2.2 F I G U R E 1 Pedigree of the described non-consanguineous Chinese family with MKS. Squares and circles denoted males and females respectively. Individuals labelled with a solidus were deceased. Roman numerals indicate generations. Arrow indicates the proband (II-6)

Funding information
The Guangdong Enterprise Key Laboratory of Human Disease Genomics, Grant/Award Number: 2011A060906007 CEP290 gene, homozygous, loss-of-function, Meckel syndrome, novel variant were used for analysing the data. The reporting threshold of copy number was set at 10 kb with marker count at ≥50. 9

| Sanger sequencing
In order to validate the identified variants by whole exome sequencing, we performed Sanger sequencing. Designing of primer pairs for the candidate loci has been done based on the reference genomic sequences of the Human Genome from GenBank in NCBI.
Primer pairs were synthesized by Invitrogen, Shanghai, China.
Polymerase chain reaction (PCR) was performed with an ABI 9700 Thermal Cycler and next, directly sequenced the PCR products by an ABI PRISM 3730 automated sequencer (Applied Biosystems).
Analysis of sequencing data has been done by DNASTAR SeqMan (DNASTAR).

| In vitro functional analysis of the novel variant by western blot
Foetal kidney tissue was rapidly frozen in liquid nitrogen and stored in ultra-low temperature freezer. The RIPA lysis buffer

| Immunofluorescence study of human kidney tissues
Foetal kidney tissue and also normal kidney tissues were fixed in 4% paraformaldehyde overnight and embedded in 4 μm thick paraffin.
Then, the glass slides were rinsed in PBS (5 minutes/time, three times) and coverslips were fixed on glass slides with fluoroshield mounting medium (containing DAPI). Confocal images were taken using a scanning microscope system (Yokogawa CSU-X1 spinning disk scanner coupled to a Zeiss Axio Imager Z2 inverted microscope and controlled by Zen Blue software).

| In silico analysis
The variant identified in the foetus by whole exome sequencing was analysed by Mutation Taster (http://mutat ionta ster.org/). 11

| Human subjects
In this present study, we investigated a 35-years-old pregnant Chinese woman who had a history of adverse pregnancy (Figure 1). This Chinese family is a truly non-consanguineous. She is 17+1-week pregnant (gravida 6, para 2). It is her 6th pregnancy and she had successful deliveries of two girl children from her previous five pregnancies. During her 6th pregnancy, she went to our hospital for routine B-ultrasound examination and we identified her foetus with multiple malformations.
During consultation, it has been revealed that she had a history of adverse pregnancy for three times among her last five pregnancies. In 2006, during her first pregnancy, she visited our hospital and  Figure 3A). The foetal kidneys were obviously enlarged, filled the entire abdominal cavity ( Figure 3B).
The size of the left kidney was about 3.0 × 2.1 × 1.9 cm, and the size of the right kidney was about 3.0 × 1.8 × 2.0 cm. There are many cystic echo regions of different sizes and shapes. Prenatal genetic diagnosis with amniocentesis at 18 weeks of gestation was performed.

| Karyotype and chromosomal microarray analyses
Karyotype analysis found no chromosomal structural abnormality in the foetus (46, XX). Chromosomal microarray did not identify any pathogenic copy number variations (CNVs) in the chromosomes of the foetus.
After identifying no abnormality in both karyotype and chromosome microarray, and based on both the multiple similar adverse pregnancy history and clinical symptoms (foetal meningocele during pregnancy and bilateral polycystic Kidney with poor prognosis) of the foetus, the pregnant woman decided to terminate the pregnancy and perform further genetic molecular diagnosis to understand the underlying cause of the disease phenotype.
We observe the aborted foetus and found occipital encephalocele ( Figure 4A), bilateral polycystic kidneys (occupying the entire fatal abdomen) ( Figure 4B,C). We have not found postaxial polydactyly in both hand and feet of the foetus ( Figure 4D,E). We also identified bilateral grossly enlarged kidneys interspersed with small, pinhead-sized cysts ( Figure 4F). Histology of kidney found cysts in kidney and thinning of renal cortex with glomerular atrophy ( Figure 4G). Histology of liver showed no abnormality ( Figure 4H).
Foetal skin tissue was taken for the extraction of genomic DNA.
Whole exome sequencing has been done with foetal genomic DNA.
F I G U R E 2 Schematic presentation of the detailed data interpretation pipeline

| Whole exome sequencing and Sanger sequencing identified a homozygous novel variant in CEP290
We performed whole exome sequencing of DNA from the skin of

| In vitro functional analysis and characterization of the novel variant by western blot
Western blot analysis showed that this variant (c.2144T>G, p.Leu715*) leads to the formation of a truncated CEP290 protein with the molecular weight of 84 KD compared with the wild-type CEP290 protein of 290 KD. In addition, the expression of the mutated CEP290 protein in foetal kidney tissue was also significantly lower in comparison with the expression of the wild-type CEP290 protein in normal kidney tissues ( Figure 6A).

| Immunofluorescence study of human kidney tissues
Immunofluorescence study showed that the expression of mutated CEP290 was quite lower in foetal polycystic kidney tissue in comparison with the expression of wild-type CEP290 protein in normal kidney tissue, which was consistent with the results of Western blot.
In addition, we also observed that the localization of the mutated CEP290 protein in foetal kidney tissues, compared to the wild-type CEP290 protein in normal kidney tissues with a magnification of 40× ( Figure 6B).
In order to understand the specific location of both wild-type and mutated CEP290 in normal as well as in foetal kidney tissues, we used high magnification (63×) immunofluorescence imaging. In accordance with previous studies, Figure 7A showed CEP290 localization to the ciliary base in normal kidney tissue. 12,13 However, CEP290 had almost no expression in foetal polycystic kidney tissue. In order to observe the effect of CEP290 variant on ciliary structure, we detected the localization and expression of ARL13B (ADP-ribosylation factor-like protein 13B) in both normal and foetal polycystic kidney tissues by immunofluorescence assay. The expression of ARL13B in foetal polycystic kidney tissues with mutated CEP290 protein was similar to the normal kidney tissues with wild-type CEP290 protein ( Figure 7B). We also found difference in the length of cilia between the wild-type and mutant kidney tissues. The mutant cilium appears longer in length than that of the wild-type cilium.
F I G U R E 5 Partial DNA sequences in the CEP290 gene by Sanger sequencing of the family. The reference sequence NM_025114.3 of CEP290 gene was used F I G U R E 6 A, Protein expression analysis using Western blotting of kidney tissue obtained from patient MKS. Lane 1 showed the CEP290 (290 KD) expression in normal kidney tissues (wild-type, WT). Lane 2 showed CEP290 expression in foetal kidney tissue (mutant type, MT). GAPDH levels, served as controls. B, Localization of CEP290 in kidney tissues of MKS patient and normal kidney tissue. DAPI, for nuclear staining (blue); CEP290, Anti-CEP290 antibody followed by an Alexa fluo488-conjugated secondary antibody (green); Merge, DAPI nuclear staining plus anti-CEP290 antibody. These images were observed using immunofluorescence microscopy (40×)

F I G U R E 7 A, Localization of CEP290
in kidney tissues of MKS patient and normal kidney tissues (63×). DAPI, for nuclear staining (blue); CEP290, Anti-CEP290 antibody followed by an Alexa Fluor 488-conjugated secondary antibody (green); Merge, DAPI nuclear staining plus anti-CEP290 antibody. B, Localization of ARL13B in kidney tissues of MKS patient and normal kidney tissues (63×). DAPI, for nuclear staining (blue); ARL13B, Anti-ARL13B antibody followed by an Alexa Fluor 488-conjugated secondary antibody (green); Merge, DAPI nuclear staining plus anti-ARL13B antibody. The white arrow indicates the cilia. These images were observed using immunofluorescence microscopy (63×)

| D ISCUSS I ON
In our present study, we described a 35-years-old pregnant Chinese woman from a non-consanguineous Chinese family. She had a history of adverse pregnancy. During her sixth pregnancy, we found that the foetus was presented with occipital meningoencephalocele with a massively malformed brain, cystic dysplastic kidneys and the pregnant woman decided to terminate the pregnancy. We  Figure 6A). Additionally, we also observed that the wild-type CEP290 protein is localized into the ciliary base in normal kidney tissue while we found no expression of mutated CEP290 protein in foetal polycystic kidney tissue ( Figure 7A). However, the expression of ARL13B in foetal kidney tissue with mutated CEP290 protein was similar to the normal kidney tissues with wild-type CEP290 protein ( Figure 7B).
In addition, here, we observed differences in the length of cilia between wild-type and mutant kidney tissues. The length of the mutant cilium has found longer than that of the wild-type cilium.
However, structural defects of cilia as well as differences in ciliary length are correlated with severe developmental diseases. 14 Polycystic kidney tissues of foetus have been suffering from MKS3 with mutation in TMEM67 gene showed longer cilia than wild-type tissues. 15 LCA patients with mutations in CEP290 gene never been identified with an increased ciliary length. Hence, the structural defects of cilia as well as differences in ciliary length are tissue specific. In addition, regulation of the length of the primary cilia is very significant and dynamic process which is playing a key role in signal transduction through cilia. 16 The structure and length of the cilia is very specific for its functional significance. 17 Germline variants in CEP290 gene rarely cause MKS4. 2

ACK N OWLED G EM ENTS
We are thankful to the proband and all the family members for participating in our study. We are also thankful to the Guangdong Enterprise Key Laboratory of Human Disease Genomics (2011A060906007).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data used for the analyses in this report are available in the CNGB Nucleotide Sequence Archive (CNSA: https ://db.cngb.org/cnsa).