Diversified clinical presentations associated with a novel sal-like 4 gene mutation in a Chinese pedigree with Duane retraction syndrome

• Introduction
• Methods
• Results
• Discussion

Abstract

Purpose: To determine the underlying genetic cause of Duane retraction syndrome (DRS) in a non-consanguineous Chinese Han family.

Methods: Detailed ophthalmic and physical examinations were performed on all members from a pedigree with DRS. All exons and their adjacent splicing junctions of the sal-like 4 (SALL4) gene were amplified with polymerase chain reaction and analyzed with direct sequencing in all the recruited family members and 200 unrelated control subjects.

Results: Clinical examination revealed a broad spectrum of phenotypes in the DRS family. Mutation analysis of SALL4 identified a novel heterozygous duplication mutation, c.1919dupT, which was completely cosegregated with the disease in the family and absent in controls. This mutation was predicted to cause a frameshift, introducing a premature stop codon, when translated, resulting in a truncated SALL4 protein, i.e., p.Met640IlefsX25. Bioinformatics analysis showed that the affected region of SALL4 shared a highly conserved sequence across different species. Diversified clinical manifestations were observed in the c.1919dupT carriers of the family.

Conclusions: We identified a novel truncating mutation in the SALL4 gene that leads to diversified clinical features of DRS in a Chinese family. This mutation is predicted to result in a truncated SALL4 protein affecting two functional domains and cause disease development due to haploinsufficiency through nonsense-mediated mRNA decay.

Duane retraction syndrome (DRS) is a congenital disorder of ocular motility, which is pan-ethnic and accounts for approximately 1% of the total cases of strabismus [1]. Individuals with DRS typically manifest horizontal eye movement limitation, globe retraction with palpebral fissure narrowing in attempted adduction. DRS can be classified into types I, II, and III, with the presence of abduction, adduction, or both, respectively [2]. Occurrence of DRS is usually sporadic. Reported familial DRS is mostly bilateral with vertical movement abnormalities and autosomal dominant inheritance [3]. In addition, several associated systemic manifestations have been described in patients with DRS, most commonly involving facial anomalies, hearing dysfunction, vertebral column anomalies, and variable degrees of limb malformations [4-7]. The exact etiology of DRS is still elusive. Electromyographic and magnetic resonance imaging (MRI) studies suggested that DRS might result from abnormal development of the abducent nerve (sixth cranial nerve) [8,9].

Linkage analyses of DRS have successfully mapped its associated loci to chromosomes 2q13, 4q27, 8q13, 22q11, and 20q13 [10-13]. Recently mutations of the sal-like 4 (SALL4; MIM# 607343) gene on chromosome 20 have been linked to DRS or DRS associated with radial forearm malformations, also known as Okihiro syndrome [14-17]. SALL4-related disorders include Duane-radial ray syndrome (DRRS, Okihiro syndrome) and acro-renal-ocular syndrome (AROS). Okihiro syndrome, characterized by radial malformation associated with Duane congenital abnormalities, is used interchangeably with DRRS. Another SALL-4 related syndrome is known as acro-renal-ocular syndrome (AROS), characterized by radial ray malformation, Duane abnormality, renal malformation (mild malrotation, ectopia, horseshoe kidney, renal hypoplasia, vesicoureteral reflux, bladder diverticula), and coloboma [14]. More than 20 mutations in SALL4 have been reported in association with these disorders. The SALL4 gene is located in chromosome 20q13–20q13.2, with a length of 18.14 kb and 4 exons, encoding for a 1053 amino-acid-residue protein. SALL4, a new member of the SAL family of proposed C2H2 zinc finger transcription factors, plays important transcription roles during human embryogenesis [15,18]. SALL4 is actually the first identified disease-causing gene of DRS and likely plays a crucial role in the development of the abducens motoneuron. Although several mutations have been reported in SALL4, disease phenotypes vary greatly among different SALL4 mutations, and the phenotype–genotype correlation remains elusive. In this paper, we report for the first time a Chinese family with members presenting with isolated DRS or DRS associated syndromes in a dominant trait.

This study was approved by the Research Ethics Committees of the Chinese University of Hong Kong and conducted in accordance with the tenets of the Declaration of Helsinki. Informed consent was obtained from each participant after detailed explanation of the nature of the study.

In this study, we identified a novel SALL4 mutation in a Chinese family with DRS presenting with variable phenotypes. The mutation showed complete disease cosegregation and is highly likely to be disease-causing, although further functional characterization is needed to confirm the mutation’s role in disease etiology. Autosomal dominant DRS has a high penetrance, although considerable variability within a pedigree also exists. This may explain the wide-spectrum phenotypes observed in the family evaluated. Until now, several sporadic cases or pedigrees and mutations implicated in DRS have been reported in Caucasians. To the best of our knowledge, this is the first genetic study, and the first pedigree report, on DRS in the Chinese population.

Mutations in the SALL4 gene have been shown to cause DRS and DRS-associated disorders, namely, Okihiro syndrome, acro-renal-ocular syndrome, Holt-Oram syndrome, or suspected thalidomide embryopathy [19-22]. Several overlapping phenotypes are shared among these disorders. Okihiro syndrome, an autosomal dominant disorder characterized by radial ray defects combined with Duane anomaly, is the most common condition known to overlap clinically with DRS. The SALL4 gene consists of four coding exons and encodes a protein with three highly conserved C2H2 double zinc finger domains, the second of which has a single C2H2 zinc finger attached at its C-terminal end, as well as an N-terminal C2HC zinc finger motif [15,18,23,24]. To date, 22 SALL4 mutations have been described in Duane-related syndromes, especially Okihiro syndrome [14-17,21,23]. Seventeen are located in exon 2, and five within exon 3. These known mutations are nonsense mutations, duplications, or deletions (Figure 7).

The c.1919dupT mutation identified in this study was a duplication located in exon 2. This mutation is predicted to generate a frameshift resulting in a truncated protein denoted as p.Met640IlefsX25. Thus, when translated, the premature termination codon would truncate the SALL4 polypeptide by 414 residues, i.e., appropriately 40% of the total length. The codon would truncate the second C2H2 double zinc finger domain and lead to a complete deletion of the third domain due to premature termination. The region containing these three functional domains was conserved; our sequence analysis also showed the deleted residues in the second domain of SALL4 to be highly conserved across different vertebrate species. This heterozygous c.1919dupT mutation plays a role in the disease pathogenesis via haploinsufficiency through a pathway known as nonsense-mediated mRNA decay, an mRNA surveillance mechanism found in all eukaryotic organisms that leads to a degradation of the transcripts with introns in the 3′ untranslated region. The mutation subsequently prevents the synthesis of truncated proteins that may have toxic effects such as dominant negative interactions [25,26]. In fact, all the mutations identified thus far in SALL4 associated with Duane-related syndromes are likely to be disease-causing via haploinsufficiency [27]. This proposition was based on the observation that disease-causing deletions of all coding exons in two independent families that explicitly resulted in haploinsufficiency [16]. The c.1919dupT mutation described in our Chinese family with DRS provides further evidence supporting that SALL4 mutations affect the disease phenotype through nonsense-mediated mRNA decay.

To date, there is insufficient information to correlate SALL4 mutations with the severity of the phenotypes of DRS and DRS-associated syndromes. Although the mutation p.R865X has been reported to cause either a mild or severe phenotype [21], c.1919dupT in this present family gives clear variations in clinical features. Varying degrees of ocular disorders and limb involvements were manifested in the c.1919dupT carriers. Such complex disease manifestation might have been modified by local epigenetics or other mutations that have yet to be identified.

Kohlhase et al. demonstrated that the expression of SALL4 in human could be detected only in the testis and ovary [18]. In animal studies, SALL4 mRNA expression has been detected in the embryo and well described during different developmental periods; the expression in the progress zone of the limb buds fitted well with the radial ray anomalies in patients with Duane-related syndromes [18]. In addition, a reduced dosage of SALL4 in the mid-hindbrain region was expected to result in disturbed development of the sixth cranial nerve [18]. Two independent studies noted no mutations in SALL4 could be detected in isolated DRS cases, suggesting other genetic influences in addition to SALL4 [28,29]. Recently, mutations in CHN1 have been found in association with isolated families with DRS [30].

In summary, we have identified a novel heterozygous SALL4 mutation cosegregating with DRS in a Chinese pedigree. This mutation is predicted to result in a truncated SALL4 protein affecting two functional domains, and haploinsufficiency due to nonsense-mediated mRNA decay may be responsible for the disease pathogenesis. The results of this study provide further evidence for understanding the genetic causes of DRS and extend its phenotypic and mutational spectra.