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Keywords:

  • cilia;
  • bromi;
  • SCLT1;
  • TBC1D32;
  • cleft;
  • polydactyly;
  • microphthalmia;
  • anophthalmia

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Clinical syndromes caused by defects in the primary cilium are heterogeneous but there are recurrent phenotypic manifestations that define them as a collective group known as ciliopathies. Dozens of genes have been linked to various ciliopathies but large patient cohorts have clearly revealed the existence of additional genetic heterogeneity, which is yet to be fully appreciated. In our search for novel ciliopathy-linked genes through the study of unmapped ciliopathy phenotypes, we have identified two simplex cases with a severe ciliopathy phenotype consistent with oro-facio-digital syndrome type IX featuring midline cleft, microcephaly, and colobomatous microphathalmia/anophthalmia. In addition, there was variable presence of polydactyly, absent pituitary, and congenital heart disease. The autozygome of each index harbored a single novel truncating variant as revealed by exome sequencing, and the affected genes (SCLT1 and TBC1D32/C6orf170) have established roles in centrosomal biology and ciliogenesis. Our findings suggest a previously unrecognized role of SCLT1 and TBC1D32 in the pathogenesis of ciliopathy in humans.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Primary cilium is a microtubule-based centrosome-derived appendage that can be detected in virtually all post-mitotic cells, and represents the best studied manifestation of the centrosome outside its role in mitosis [Eggenschwiler and Anderson, 2007]. Numerous biological functions have been assigned to the primary cilium including determination of cell polarity and serving as mechanosesnor and mediator of important signaling cascades particularly Sonic Hedgehog (SHH) [Nozawa et al., 2013]. Not surprisingly, defects in this ubiquitous cellular organelle adversely affect cellular homeostasis in a pattern that manifests systemically. Ciliopathies are clinical conditions that share in common abnormal ciliary function and/or structure, and have been classified into distinct syndromes each with its unique constellation of clinical findings [Waters and Beales, 2011]. The recurrent presence of certain phenotypic aspects (e.g., polydactyly, retinal dystrophy, polycystic kidneys) in apparently disparate syndromes has long hinted to a shared molecular mechanism but it was the recent revelation of mutations in dozens of cilia-related genes that made it possible to view these syndromes as a collective group that came to be known ciliopathies [Quinlan et al., 2008]. The tendency to view these groups collectively is further illustrated by the demonstration of numerous examples where mutations in the same gene can result in more than one clinically recognizable syndrome [Abdelhamed et al., 2013; Lee and Gleeson, 2011].

Despite the identification of dozens of genes that are mutated in patients with various ciliopathies, the rapidly accumulating literature on the numerous proteins that play critical roles in the various aspects of ciliary biology strongly suggests that the disease genes identified to date only represent a fraction of what the entire set of cilia-related genes (ciliome) actually contributes to human disease [Kim et al., 2010]. Indeed, in none of known ciliopathies has the entire genetic heterogeneity been captured although the contribution of known disease genes ranges widely between various disorders. For example, while the majority of patients with Bardet–Biedl syndrome, Joubert syndrome, and Meckel–Gruber syndrome harbor mutations in known genes, most cases of non-X-linked Oro-Facio-Digital (OFD) syndrome cannot be traced to mutations in known genes [Abu-Safieh et al., 2012; Alazami et al., 2012; Shaheen et al., 2013b].

Identification of ciliopathy-related genes has historically relied on positional mapping strategies that employ linkage analysis and autozygosity mapping, and while this method can be very powerful, it is often limited by the availability of suitable pedigrees. Candidate gene analysis approach has also been successful in the setting of ciliopathies as nearly the entire compendium of the ciliome is known [Chen et al., 2006], but the very large number of candidates and lack of knowledge about which of these will be clinically consequential greatly reduces the efficiency of this method. Next-generation sequencing has markedly accelerated novel gene discovery by empowering both approaches mentioned above to fully harness their strengths and overcome their major limitations. For example, it is now possible to identify causal mutations in simplex ciliopathy cases [Shaheen et al., 2013a] and it is also possible to multiplex-sequence a large number of ciliome candidate genes in a large cohort of patients with various ciliopathies [Hopp et al., 2011]. In this study, we report the successful use of whole-exome sequencing of two simplex cases with a severe ciliopathy phenotype to identify truncating mutations in two genes that have not been previously linked to ciliopathy in humans despite their established role in various aspects of ciliary biology.

Materials and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Human Subjects

Patients and their relatives were recruited using a KFSHRC IRB-approved protocol (RAC# 2080006) with informed consent. Venous blood was collected in EDTA and PAXGene tubes for DNA and RNA extraction, respectively.

Autozygome Analysis

The entire set of autozygous intervals per individual (autozygome) was determined for each index as described before [Alkuraya, 2012]. Briefly, extracted genomic DNA was genotyped on the Axiom SNP Chip platform following the manufacturer's instructions (Affymetrix, Santa Clara, CA) followed by determination of homozygous intervals as surrogates of autozygosity using AutoSNPa [Carr et al., 2006].

Whole-Exome Sequencing

Standard whole exome sequencing was performed using TruSeq Exome Enrichment kit (Illumina, San Diego, CA, USA) followed by preparation of an Illumina sequencing library, with enrichment for the desired target using the Illumina Exome Enrichment protocol. The captured libraries were sequenced using Illumina HiSeq 2000 Sequencer. The reads were mapped against UCSC hg19 by BWA. The SNPs and Indels were detected by SAMTOOLS. Only coding/splicing variants that are present within the autozygome and are novel were considered as candidate variants as described [Alkuraya, 2013].

RTPCR

To confirm the predicted effect of splicing mutations, RNA was extracted using the Qiagen RNeasy Mini Kit (Valencia, CA), treated with RNase-free DNase (Qiagen), then converted into cDNA (Reverse Transcription System; Promega, Fitchburg, WI) which was used as a template for subsequent PCR. Primers that span exon–exon junctions were used to avoid inadvertent genomic DNA amplification. Primers and conditions are available upon request.

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Identification of Two Patients with a Severe Ciliopathy Phenotype

Index 1 is a male born to healthy consanguineous Saudi parents. He was found antenatally at 24 weeks of gestation to have cleft lip, abnormal hands, and small occipiofrontal circumference. At birth, he was noted to have microcephaly, right microphthalmia, left anophthalmia, bilateral optic disc coloboma, severe midline cleft (lip and alveolus), hypertelorism, severe choanal stenosis, left hand post-axial polydactyly, and ambiguous genitalia (Fig. 1). Echocardiogram revealed the presence of patent ductus arteriosus (PDA) and atrial septal defect (ASD). Soon after birth, he developed panhypopituitarism secondary to absent pituitary gland as confirmed by brain MRI, which also showed partial agenesis of the corpus callosum and agenesis of the inferior cerebellar vermis. Abdominal and renal ultrasounds were normal. Computed tomography (CT) of the nasal cavity and sinuses reported narrow choanal opening and thickening of the anterior aspect of nasal cavity. In the neonatal intensive care unit (NICU), he developed tonic clonic seizures, and EEG showed focal discharge from the right parietotemporal area. He developed severe electrolyte imbalance and died at 6 months of age with cardiac arrest.

image

Figure 1. Pedigrees of the study families and representative images of each proband.

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Index 2 is another male born to healthy consanguineous Saudi parents, unrelated to the first family. Similar to Index 1, he had a severe midline cleft lip and palate, microcephaly and choanal atresia (Fig. 1). He also had significant eye involvement in the form of severe coloboma, and congenital heart disease (ASD and VSD) but his genitalia were less severely affected than Index 1 with just micropenis. Brain imaging revealed pachygyria and absent corpus callosum. Unlike Index 1, he had abnormal inner ear structures. Patient required oxygen supplementation by nasal cannula until the age of 2 months when he developed severe respiratory tract infection necessitating mechanical ventilation but he developed cardiorespiratory arrest and died at 3 months of age. The key clinical features of both patients are summarized in Table 1.

Table 1. Clinical Characteristics of Two Patients with OFD IX
 Index 1Index 2
  1. PDA, patent ductus ateriosus; CHD, congenital heart disease; ASD, atrial septal defect; VSD, ventricular septal defect; ACC, agenesis of corpus callosum.

Age6 m (age at death)3 m (age at death)
GenderMaleMale
CLPLip and alveolus (midline)Lip and palate (midline)
Microcephaly++
Microphthalmia/anophthalmiaBilateral
ColobomaBilateralBilateral
PolydactylyLeft hand
Abnormal genitalia++
Choanal stenosis/atresia++
CHDPDA and ASDASD and VSD
Brain MRIACC, absent pituitary and agenesis of inferior vermisACC, pachygyria, and abnormal inner ear
Seizures+

Exome Sequencing Reveals Truncating Mutations in TBC1D32 and SCLT1

We hypothesized that the cause of ciliopathy in both patients is a homozygous mutation that was inherited on an autozygous background given the consanguineous nature of their parents, but that the underlying gene may or may not be the same. Indeed, the autozygome of the two probands did not show any overlap suggesting locus heterogeneity for this condition, so we proceeded with exome sequencing of each proband, and the variants were analyzed independently. By only considering novel coding/splicing variants within the autozygome of each proband, only one variant survived filtration in each case (Supp. Fig. S1). In Index 1, a splicing mutation was identified in C6orf170 (NM_152730.4:exon12:c.1372+1G>T) that is predicted to fully abolish the consensus donor site downstream of exon 12. Indeed, RTPCR confirmed complete skipping of exon 12 resulting in in-frame truncation of 47 amino acids (p.Arg411_Gly458del) (Fig. 2). Similarly, the only variant that survived filtration in Index 2 was a splicing mutation in SCLT1 (MIM #611399, NM_144643.2:exon5:c.290+2T>C). This mutation completely abolishes the consensus donor site of exon 5 as confirmed by RTPCR, which showed complete skipping of exon 5 resulting in a frameshift and introduction of a premature stop codon (p.Lys79Valfs*4), but there was no evidence of NMD (Fig. 3 and data not shown). Neither mutation was found in 250 in-house Saudi exomes, or by Sanger sequencing of 96 Saudi controls. In addition, neither variant is present in dbSNP, 1000 Genomes or Exome Variant Server. The variants have been submitted to their respective gene-specific variant databases www.lovd.nl/TBC1D32 and www.lovd.nl/SCLT1.

image

Figure 2. Upper panel: Schematic of TBC1D32/C6orf170 with the site of the mutation denoted by a red star. Lower panel: Sequence chromatogram of the aberrant transcript recovered from patient blood as compared with control.

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image

Figure 3. Upper panel: Schematic of SCLT1 with the site of the mutation denoted by a red star. Lower panel: Sequence chromatogram of the aberrant transcript recovered from patient blood as compared with control.

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Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

In this study, we identify, to the best of our knowledge, the first human variants in TBC1D32/C6orf170 and SCLT1 in two patients with a severe ciliopathy phenotype. Midline cleft and polydactyly are frequently encountered features in OFD. Microcephaly, cortical dysgenesis, absent corpus callosum, microphthalmia, coloboma, and absent pituitary have also been reported in patients with OFD. Therefore, the ciliopathy phenotype we describe best fit OFD, specifically OFD type IX in view of the prominent eye involvement although we note the lack of classic tongue involvement [Gurrieri et al., 2007]. The genetics of this subtype is unknown. The mutations we identified in C6orf170 and SCLT1 are highly interesting because they represent the only variants that survived filtration in each case, and because of the recent literature that assigned a critical role to each of these two genes in the primary cilium.

C6orf170 (HGNC-approved gene symbol TBC1D32) encodes a protein predicted to contain a Tre-2, Bub2, and Cdc16 (TBC) domain (TBC1D32). This protein was of unknown function until a recent study mapped a mouse mutant known as bromi to the mouse ortholog of C6orf170 [Ko et al., 2010]. bromi (Broad-Minded) is an ENU-mutagenesis derived mouse line that is characterized by abnormal brain development, severe microphthalmia and polydactyly, features highly reminiscent of the phenotype observed in Index 1 [Zohn et al., 2005]. Ko et al. (2010) have shown that lack of this protein leads to grossly abnormal cilia in which large empty spaces are formed on one side of the axoneme in a pattern that suggests abnormal coordination of axoneme and ciliary membrane growth. Indeed, Ko and colleagues have also shown that TBC1D32 interacts directly with CCRK (MIM #610076), the only known mammalian ortholog of LF2p which is a component of the cytoplasmic length regulatory complex, and that ccrk knockdown recapitulates the ciliary phenotype observed in bromi morphant zebrafish [Ko et al., 2010]. More recently, proteomic analysis of the cilia has revealed that TBC1D32 is indeed a ciliary protein [Ishikawa et al., 2012]. So while the exact role of TBC1D32 in the cilia is unknown, it is clear that it is a ciliary protein deficiency of which causes abnormal ciliary development with consequent impaired SHH signaling in addition to causing a ciliopathy phenotype in mouse and zebrafish. Therefore, although the effect of the mutation we identified on C6orf170 is unknown, it is likely to play a role in the disease pathogenesis.

The case for a potentially causal role of the frameshift in SCLT1 is also strong. This gene was identified very recently as an essential component of the distal appendages, a centrosomal extension that establishes the connection between the mother centriole and the plasma membrane [Tanos et al., 2013]. Specifically, Tanos et al. (2013) identified SCLT1 along with four other proteins by performing subtraction proteomic analysis of the mother centriole and daughter centriole taking advantage of the fact that only the mother centriole has the capacity to form distal appendages. In a series of elegant experiments, they were able to establish SCLT1, just like the other proteins they identified, as being necessary for ciliogenesis and that its deficiency blocks ciliogenesis at the earliest known stage i.e. docking of the mother centriole to the plasma membrane, even before the establishment of the transitional zone [Tanos et al., 2013]. Although the active domains of SCLT1 in this novel role have not been mapped, we note that our patient's mutation knocks out 609 amino acids of this 688 amino-acid protein so it is likely to be null and that it played a role in the pathogenesis of this ciliopathy phenotype. However, we cannot rule out the possibility that the splicing mutation may spare one or more transcriptional functional isoforms that may render the mutation hypomorphic instead.

Despite the rarity of OFD IX, we show that it is genetically heterogeneous as evident by the autozygosity analysis of two patients born to consanguineous parents. With the exception of OFD I, which is caused by mutation in OFD1 (MIM #300170) which we excluded in these two patients by sequencing, all other OFD subtypes are autosomal recessive and while several disease genes have been identified (TMEM216; MIM #613277, TCTN3; MIM #613847) and most recently DDX59 (MIM #615464) [Shamseldin et al., 2013; Thomas et al., 2012; Valente et al., 2010]), the majority of patients have no molecular diagnosis. Our results suggest that the two ciliary genes TBC1D32/C6orf170 and SCLT1 are likely candidates that should be considered in the mutation analysis of autosomal recessive OFD. Since the bottleneck in Mendelian disease gene identification has quickly shifted from mapping to independent confirmation, we caution that identifying mutations in these two genes in future patients will be necessary to establish these two genes as bona fide ciliopathy genes in humans rather than a recently recognized group of genes that seem to tolerate biallelic loss of function in humans [MacArthur et al., 2012].

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

We thank the families for their enthusiastic participation. We also thank the Genotyping and Sequencing Core Facilities at KFSHRC for their technical help.

Disclosure statement: The authors declare no conflict of interest.

References

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

FilenameFormatSizeDescription
humu22477-sup-0001-FigureS1.pdf163KFigure S1. Filtering strategy used to analyze the whole-exome variants in the study patients. The number of variants that remain after each filter are shown in the corresponding circle. The color code is shown to the right.

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