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Lab Med Online. 2017 Jan;7(1):45-48. English.
Published online Jan 01, 2017.  https://doi.org/10.3343/lmo.2017.7.1.45
© 2017, Laboratory Medicine Online
Whole Exome Sequencing in a Korean Child with Joubert Syndrome-related Disorders
Jong Hwa Lee, M.D.,1,5 In Kyung Oh, M.D.,2 Mi Jin Yoon, M.D.,3 and Kui Hyun Yoon, M.D.4,5
1Department of Pediatrics, Wonkwang University Sanbon Hospital, Gunpo, Korea.
2Department of Ophthalmology, Wonkwang University Sanbon Hospital, Gunpo, Korea.
3Department of Radiology, Wonkwang University Sanbon Hospital, Gunpo, Korea.
4Department of Laboratory Medicine, Wonkwang University Sanbon Hospital, Gunpo, Korea.
5Institute of Wonkwang Medical Science, Iksan, Korea.

Corresponding author: Kui Hyun Yoon. Department of Laboratory Medicine, Wonkwang University Sanbon Hospital, 321 Sanbon-ro, Gunpo 15865, Korea. Tel: +82-31-390-2658, Fax: +82-31-391-2085, Email: wooju67@paran.com
Received March 31, 2016; Revised June 16, 2016; Accepted June 21, 2016.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract

Joubert syndrome and Joubert syndrome-related disorders (JSRDs) are rare autosomal recessive or X-linked disorders characterized by cerebellar vermis hypoplasia and a brain stem malformation, which presents as the “molar tooth sign” in magnetic resonance imaging (MRI). JSRDs are a group of clinically heterogeneous conditions that exhibit neurological manifestations and multiple organ involvement. JSRDs are also genetically heterogeneous, and approximately 20 causative genes that account for 45% of JSRDs have been identified. A 7-yr-old boy visited Wonkwang University Sanbon Hospital with the following presentations: no ocular fixation, ataxia, growth retardation, and hypotonia. Physical examination revealed facial dysmorphism, spindle shaped fingers, and height (99 cm) and weight (13 kg) below the third percentile. Ophthalmic examination revealed retinal dystrophy. A diagnosis of JSRDs was made based on clinical and brain MRI findings. We found two heterozygous variants c.2945 G>T; p.Arg982Met (G>T) and c.2216dupA; p.Phe740Valfs*2 (dupA) in AHI1, and a heterozygous c.3973C>T; p.Arg1325Trp (C>T) variant in KIF7 by whole exome sequencing (WES). Genetic analysis on the proband's father revealed that he had both AHI1 variants, but did not have the KIF7 variant, which was inconsistent with autosomal recessive inheritance. Therefore, the G>T variant and C>T variant were presumed to be of “uncertain significance.” Furthermore, one novel dupA variant was interpreted as “pathogenic,” while the second allele was not detected. Caution should be exercised while interpreting the significance of variants detected by WES. In addition, the involvement of genes other than the 20 known ones will require further investigation to elucidate the pathogenesis of JSRDs.

Keywords: Joubert syndrome; Whole exome sequencing; AHI1; KIF7


Joubert syndrome (JS) and Joubert syndrome-related disorders (JSRDs) are rare autosomal recessive or X-linked disorders. Their characteristic features include cerebellar vermis hypoplasia and a brain stem malformation that presents as the diagnostic marker “molar tooth sign” in magnetic resonance imaging (MRI). JSRDs are clinically heterogeneous, showing neurological manifestations 6338and multiple organ involvement, particularly of the retina, kidney, liver, and skeleton. Therefore, they are classified into six subtypes: pure JS, JS with ocular defect, JS with renal defect, JS with oculorenal defects, JS with hepatic defect, and JS with orofaciodigital defects [1]. JSRDs are also genetically heterogeneous, and approximately 20 causative genes, accounting for 45% of JSRDs have been identified [2]. However, the number of identified genes is likely to increase with the discovery of novel genes [2, 3] (Table 1). More so since the diagnostic value of next generation sequencing in rare inheritance disorders has been recently reported [4, 5, 6].

The patient was a 7-yr-old boy, who visited Wonkwang University Sanbon Hospital with the principal presentations of lack of ocular fixation, ataxia, growth retardation, and hypotonia. Physical examination revealed facial dysmorphism and spindle shaped fingers, while ophthalmic examination revealed retinal dystrophy. Furthermore, his height (99 cm) and weight (13 kg) were below the third percentile. JSRDs was diagnosed based on clinical and brain MRI findings (Fig. 1). Routine hematological and biochemical analyses were within normal limits (e.g., bilirubin, AST, ALT, blood urea nitrogen, creatinine), and his chest X-ray and abdominal CT findings were normal. His family history was unremarkable.


Fig. 1
Brain magnetic resonance imaging showing the typical “molar tooth sign” attributed to cerebellar vermis hypoplasia and brainstem malformation.
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A few cases of JSRDs have been reported in Korea, diagnosed based on clinical and radiological findings, but without any molecular genetic studies [7, 8, 9]. To identify causative mutations, whole exome sequencing (WES) was performed with the patient's DNA (with the written informed consent of the proband's father) and the 20 known causative genes of JSRDs were included in the analysis [2]. Genomic DNA was enriched using the SureSelect all exon V4 (Agilent Technologies, Santa Clara, CA, USA), which targets 334,378 exons of a 51 Mb region spanning 20,965 genes. WES was performed using an Illumina HiSeq 2000 (Illumina Inc., San Diego, CA, USA) with the reference sequence UCSC assembly hg19 (http://genome.ucsc.edu/) and the BWA mapping program (http://bio-bwa.sourceforge.net/). SNPs and indels were detected using SAMTOOLS (http://samtools.sourceforge.net/). A mean coverage of 101.0X was achieved and 98.3% of targeted paired-end sequences were read more than 10 times by exome capture and sequencing. A total of 69,157 SNPs were identified and pathogenic variants were prioritized as follows [10]. Initially, the 20 known causative genes of JSRDs were selected as a target for analysis. Of the 23 exonic variants, 8 synonymous variants and 10 variants with allele frequency of ≥0.05 in the 1000 Genomes Project (http://1000genomes.org) were excluded, which left five candidate variants. These variants were not been previously reported in the 1000 Genomes Project. The first variant was a heterozygous c.6860G>A; p.Ser2287Asn in C5orf42 (NM_023073.3), which was predicted to be tolerated and benign by SIFT and PolyPhen at Ensembl Genome Browser (http://ensembl.org). Its variant frequency was 0.0113 in the Korean Reference Genome Database (KRGDB) (http://152.99.75.168/KRGDB/menuPages/rstInfo.jsp), which corresponds to a polymorphism. In addition, a heterozygous c.501G>T; p.Lys167Asn variant in CC2D2A (NM_001080522.2) and a heterozygous c.3973C>T; p.Arg1325Trp (C>T) variant in KIF7 (NM_198525.2) were predicted to be probably damaging by PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/index.shtml). The frequencies of these variants were 0.0129 and 0.0008, respectively, in the KRGDB. We excluded CC2D2A variant because of polymorphism. The heterozygous C>T variant in KIF7 was considered to be of uncertain significance rather than a primary pathogenic cause because JSRDs with this gene variant is inherited in an autosomal recessive manner and the other variant allele was not detected. Two heterozygous variants, that is, c.2945G>T; p.Arg982Met (G>T) and c.2216dupA; p.Phe740Valfs*2 (dupA) in AHI1 (NM_017651.4) were considered possible pathogenic variants. The frequency of the G>T variant was 0.0040 in KRGDB, but the dupA variant was not present in the 1000 Genomes Project or the KRGDB. These variants occurring between exon 13 and exon 20 of AHI1 were expected to lose the WD 40 domain and SH3 domain at the C-terminus of the AHI1 protein thus damaging it [11, 12]. These candidate variants were confirmed by Sanger sequencing using an ABI PRISM 3730XL Analyzer (Applied Biosystems Inc., Foster, CA, USA) (Fig. 2). We found two heterozygous variants G>T and dupA in AHI1, and a heterozygous C>T variant in KIF7 in a JSRDs patient using WES. Although we were unable to perform genetic analysis on the proband's mother, his father had both AHI1 variants, but not the KIF7 variant, which was inconsistent with autosomal recessive inheritance. Therefore, the G>T variant in AHI1 and C>T variant in KIF7 were presumed to be of “uncertain significance.” One novel dupA variant in AHI1 was interpreted “pathogenic,” and its second allele may be located in noncoding regulatory or deep intronic regions that cannot be detected by WES. AHI1 variants in JSRDs are known to be associated with risks of developing retinal dystrophy and kidney disease [11]. KIF7 variants are implicated in craniofacial dysmorphism and epiphyseal dysplasia [13].


Fig. 2
Sequencing data from the variant alleles: (A) c.3973C>T (C>T) variant of KIF7 and (B) c.2945G>T (G>T) and c.2216dupA (dupA) variants of AHI1 were confirmed by Sanger sequencing in the proband and his father.
Abbreviations: w/t, wild type; NT, no test.
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WES has the potential to become an effective tool for the diagnosis of rare heterogeneous genetic disorders because of its capacity to sequence several genes simultaneously. However, caution should be exercised when interpreting the significance of the variants identified by WES. In addition, genes other than the 20 known ones should be further investigated to fully elucidate the pathogenesis of JSRDs.

Notes

This article is available from http://www.labmedonline.org

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST:No potential conflicts of interest relevant to this article were reported.

ACKNOWLEDGMENTS

This study was supported by the research fund of Wonkwang University (2016).

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