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AIMS Primary mediastinal germ cell tumours (PMGCTs) are rare mediastinal neoplasms, and their diagnosis can be challenging, owing to small biopsy samples. The aim of this study was to develop a diagnostic algorithm using immunohistochemical staining, with a focus on novel markers, and molecular analysis of isochromosome 12p [i(12p)]. METHODS AND RESULTS Paraffin-embedded tissues of 32 mediastinal tumours were analysed with immunohistochemical staining for sal-like transcription factor 4 (SALL4), Lin-28 homologue A (LIN28), octamer-binding transcription factor 3/4 (OCT3/4), D2-40, cluster of differentiation 117 (CD117), sex-determining region Y-box 17, sex-determining region Y-box 2 (SOX2), cluster of differentiation 30, the β-subunit of human chorionic gonadotropin (β-hCG), GATA-binding protein 3 (GATA3), forkhead box protein A2 (FOXA2), glypican-3 (GPC3), α-fetoprotein (AFP), terminal deoxynucleotidyl transferase (TdT), nuclear protein of the testis (NUT), and pan-cytokeratin. Quantitative real-time polymerase chain reaction was performed to investigate the i(12p) status. Fifteen seminomas, seven teratomas, one yolk sac tumour, one choriocarcinoma and seven mixed PMGCTs were diagnosed. Each entity had different immunohistochemical staining patterns, which helped to distinguish them: OCT3/4, D2-40, CD117 and TdT for seminoma; OCT3/4 and SOX2 for embryonal carcinoma; FOXA2, GPC3 and AFP for yolk sac tumour; and β-hCG and GATA3 for choriocarcinoma. Mature teratomas stained positively for pan-cytokeratin in epithelial components and focally for SALL4, SOX2, GATA3, D2-40, and FOXA2. Furthermore, a NUT carcinoma mimicking a PMGCT was diagnosed, showing strong nuclear SOX2 staining and speckled nuclear NUT staining. i(12p) was detected in 24 of 27 PMGCTs (89%). CONCLUSION A diagnostic algorithm is of great importance for a reliable diagnosis of PMGCT in, usually small, tissue biopsy samples. Therefore, a combination of three to four antibodies to identify the correct histological subtype is usually necessary, in addition to morphological features. The i(12p) status serves as an additional option to indicate a germ cell origin in selected cases.

SALL4 is overexpressed in many cancers and is found to be involved in tumorigenesis and tumor progression. However, the function of SALL4 in cervical cancer remains unknown. Here, we showed that the expression of SALL4 was gradually increased from normal cervical tissue to high‐grade squamous intraepithelial lesions and then to squamous cervical carcinoma. SALL4 was upregulated or downregulated in cervical cancer cells by stably transfecting a SALL4‐expressing plasmid or a shRNA plasmid targeting SALL4, respectively. In vitro, cell growth curves and MTT (3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assays showed that SALL4 promoted the cell proliferation of cervical cancer cells. In vivo, xenograft experiments verified that SALL4 enhanced the tumor formation of cervical cancer cells in female BALB/c Nude mice. Cell cycle analysis by fluorescence‐activated cell sorting found that SALL4 accelerates cell cycle transition from the G0/G1 phase to the S phase. TOP/FOP‐Flash reporter assay revealed that SALL4 significantly upregulates the activity of Wnt/β‐catenin pathway. Western blotting showed that the expression levels of β‐catenin and important downstream genes, including c‐Myc and cyclin D1, were increased by SALL4 in cervical cancer cells. Furthermore, dual‐luciferase reporter and chromatin immunoprecipitation assays confirmed that SALL4 transcriptionally activated CTNNB1 by physically interacting with its promoters. Taken together, The results of this study demonstrated that SALL4 may promote cell proliferation and tumor formation of cervical cancer cells by upregulating the activity of the Wnt/β‐catenin signaling pathway by directly binding to the CTNNB1 promoter and trans‐activating CTNNB1.

Zinc-finger protein 384 (ZNF384) fusion (Z-fusion) genes have recently been identified as recurrent fusion genes in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) and have been detected in 7-17% of Philadelphia chromosome-negative BCP-ALL cases. We selected SALL4 and ID2 as potential Z-fusion-specific transcriptional targets that might lead to the differentiation disorder of Z-fusion-positive ALL. The introduction of EP300-ZNF384 and SYNRG-ZNF384 induced the expression of these genes. Z-fusion proteins exhibited stronger transcriptional activities on the promoter or enhancer region of these genes than Wild-Z. Furthermore, GST pull-down assay revealed that Z-fusion proteins associated more strongly with EP300 than Wild-Z. Coexpression of EP300 specifically enhanced the transcriptional activities of Z-fusion proteins. We propose the increased EP300 binding of Z-fusion proteins as a mechanism for their increased transcriptional activities.

Radiotherapy is the mainstay and primary curative treatment modality in nasopharyngeal carcinoma (NPC), whose efficacy is limited by the development of intrinsic and acquired radioresistance. Thus, deciphering new molecular targets and pathways is essential for enhancing the radiosensitivity of NPC. SALL4 is a vital factor in the development and prognosis of various cancers, but its role in radioresistance remains elusive. This study aimed to explore the association of SALL4 expression with radioresistance of NPC. It was revealed that SALL4 expression was closely correlated with advanced T classification of NPC patients. Inhibition of SALL4 reduced proliferation and sensitized cells to radiation both in vitro and in vivo. Furthermore, SALL4 silencing increased radiation‐induced DNA damage, apoptosis, and G2/M arrest in CNE2 and CNE2R cells. Moreover, knockdown of SALL4 impaired the expression of p‐ATM, p‐Chk2, p‐p53, and anti‐apoptosis protein Bcl‐2, while pro‐apoptosis protein was upregulated. These findings indicate that SALL4 could induce radioresistance via ATM/Chk2/p53 pathway and its downstream proteins related to apoptosis. Targeting SALL4 might be a promising approach for the development of novel radiosensitizing therapeutic agents for radioresistant NPC patients.

Primary signet-ring stromal tumour of the testis (PSRSTT) is a very rare type of primary testicular tumour, which is not actually mentioned in the current WHO categorization of tumours of the male genital organs. In this case we report about histologically and immunohistochemically features of this rare tumour. In the present case, PSRTT was found in the orchiectomy preparation of a 69-year-old man with painless swelling of his left testicle. The tumour was well circumscribed and separated from the normal testicular tissue by a fibrous capsule. The tumour cells showed a signet-ring pattern and were separated by thin collagen bundles. No mitotic activity and necrosis were found inside the tumour. Immunohistochemically, the tumour was positive for vimentin, neuron-specific enolase (NSE), S-100 protein, β-catenin and cyclin D1. No expression of cytokeratin, actin, SALL4, OCT3/4, and especially, inhibin and calretinin were detected. In conclusion, the presence of signet-ring cell formation can be found in different tumours of the testis. PSRSTT is a rare tumour that needs to be considered, in particular, to distinct them from other tumours with signet-ring cell pattern.

Breast cancer is the most prevalent cancers worldwide and causes a significant amount of deaths annually. Spalt-like transcription factor 4 is known as a transcription factor, which has an important role in the proliferation of cancerous cells. Small interfering RNA (siRNA) is a short-chain molecule of 20 to 25 nucleotides that protrude on two sides of the 3', two nucleotides. In this study, using a specific sequence of siRNA against the sequence of this gene, its activity is investigated in the cell line of breast cancer. The breast cancer cells (MCF-7) were cultured and then, using a specific anti-sal-like 4 (SALL4) siRNA, their toxic doses were determined. Then, the gene is transfected into the cell. Proliferation and expression of the SALL4 and BCL-2 gene were measured using the real-time polymerase chain reaction method. The specific concentration of siRNA IC50 of the SALL4 gene was 40.35 nmole. Gene expression results indicated that the expression of the Bcl-2 gene in the siRNA group was significantly reduced ( P < 0.05). SiRNA can increase the apoptosis of breast cancer cells by reducing the gene expression of SALL4 gene and Bcl-2; it can be used as a novel targeted therapy. This strategy, in addition to increasing the specificity of the drug, also reduces the side effects when compared with conventional chemotherapy.

Understanding the mechanism by which cancer cells enhance stemness facilitates cancer therapies. Here, we revealed that a stem cell transcription factor, SALL4, functions to enhance stemness in basal‐like breast cancer cells. We used shRNA‐mediated knockdown and gene overexpression systems to analyze gene functions. To evaluate stemness, we performed a sphere formation assay. In SALL4 knockdown cells, the sphere formation ability was reduced, indicating that SALL4 enhances stemness. CD44 is a membrane protein and is known as a stemness factor in cancer. CD44 splicing variants are involved in cancer stemness. We discovered that SALL4 modulates CD44 alternative splicing through the upregulation of KHDRBS3, a splicing factor for CD44. We cloned the KHDRBS3‐regulated CD44 splicing isoform (CD44v), which lacks exons 8 and 9. CD44v overexpression prevented a reduction in the sphere formation ability by KHDRBS3 knockdown, indicating that CD44v is positively involved in cancer stemness. In addition, CD44v enhanced anoikis resistance under the control of the SALL4 ‐ KHDRBS3 network. Basal‐like breast cancer is an aggressive subtype among breast cancers, and there is no effective therapy so far. Our findings provide molecular targets for basal‐like breast cancer therapy. In the future, this study may contribute to the establishment of drugs targeting cancer stemness.

Cranial nerves innervate head muscles in a well-characterized and highly conserved pattern. Identification of genes responsible for human congenital disorders of these nerves, combined with the analysis of their role in axonal development in animal models, has advanced understanding of how neuromuscular connectivity is established. Here, we focus on the ocular motor system, as an instructive example of the success of this approach in unravelling the aetiology of human strabismus. The discovery that ocular motility disorders can arise from mutations in transcription factors, including HoxA1, HoxB1, MafB, Phox2A, and Sall4, has revealed gene regulatory networks that pattern the brainstem and/or govern the differentiation of cranial motor neurons. Mutations in genes involved in axon growth and guidance disrupt specific stages of the extension and pathfinding of ocular motor nerves, and have been implicated in human strabismus. These genes encompass varied classes of molecule, from receptor complexes to dynamic effectors to cytoskeletal components, including Robo3/Rig1, Alpha2-chimaerin, Kif21A, TUBB2, and TUBB3. A current challenge is to understand the protein regulatory networks that link the cell surface to the cytoskeleton and to dissect the coordinated signalling cascades and motile responses that underpin axonal navigation. Here we review recent insights derived from basic and clinical science approaches, to show how, by capitalising on the strengths of each, a more complete picture of the aetiology of human congenital cranial dysinnervation disorders can be achieved. This elucidation of these principles illustrates the success of clinical genetic studies working in tandem with molecular and cellular models to enhance our understanding of human disease.

Aims To investigate the expression of spalt-like transcription factor 4 (SALL4), a regulator of embryonal development, in three epithelial components of hepatoblastoma (HB) and the relationship between SALL4 expression levels and patients' clinicopathological features. Methods and results A total of 115 specimens from 79 patients with HB were selected for immunostaining of SALL4. Nuclear staining was semi-quantified using the immunoreactive score (IS; range: 0-12). SALL4 expression was seen in all embryonal components (mean IS = 8.58) and in 41% of fetal components (mean IS = 0.78). No SALL4 expression was seen in either small cell undifferentiated or mesenchymal components of HB. Neither chemotherapy nor metastasis altered SALL4 expression significantly. High SALL4 expression levels were associated significantly with decreased overall survival (OS) (P = 0.004), event-free survival (EFS) (P = 0.003) and the presence of metastasis (P = 0.049) on univariate analysis. Multivariate analysis identified SALL4 as an independent prognostic predictor for OS (P = 0.029). Conclusions SALL4 is useful for subtyping HB, and high SALL4 expression is associated with decreased survival in HB.

Aim Combined hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) (cHCC-CC) is a rare biphasic liver cancer. Recent studies have demonstrated that cHCC-CC originates from hepatic progenitor cells (HPCs). Spalt-like transcription factor 4 (SALL4) is a marker for a progenitor subclass of HCC with an aggressive phenotype. However, little has been revealed about SALL4 expression in cHCC-CC. The aims of this study were to report SALL4 immunopositivity and the results of clinicopathological analysis in cHCC-CC, and to examine the two different nuclear immunostaining patterns for SALL4. Methods and results We defined the diffuse finely granular nuclear immunostaining pattern as immunopositive for SALL4; this was observed in eight (8.9%) of 90 cHCC-CCs. SALL4 immunopositivity was significantly associated with immunopositivity for α-fetoprotein, glypican 3, and epithelial cell adhesion molecule (EpCAM). There was no relationship between SALL4 immunopositivity and prognosis. We confirmed SALL4 mRNA expression in samples with a punctuate/clumped immunostaining pattern, which showed a significantly lower rate of immunopositivity for EpCAM than those with a diffuse finely granular pattern. Conclusions SALL4 immunopositivity is not a prognostic factor in cHCC-CC; however, it is associated with α-fetoprotein, glypican 3 and EpCAM immunopositivity, indicating the mechanism of carcinogenesis. Further study is necessary to interpret the immunostaining pattern for SALL4.

Background: Mammalian first lineage segregation generates trophectoderm (TE) and pluripotent inner cell mass (ICM), which provides an ideal model for studying the mechanisms of maintenance and loss of pluripotency. In mouse, the transcription factor OCT4 restricts to ICM and plays a key role in TE/ICM specification and pluripotent regulatory networks. However, in pig, OCT4 does not restrict to ICM cells, suggesting a different molecular basis in TE/ICM specification and pluripotent regulatory networks. Results: To explore molecular basis of porcine TE/ICM specification and pluripotent regulatory networks, we examined expression pattern of pluripotency factors, including SOX2, REX1, SALL4, ESG1, NANOG, TBX3, LIN28, KLF2, and KLF5, in porcine blastocysts. We found that SOX2 is a faithful pluripotent marker that anchored to the pluripotent cells including embryonic part cells, ICM cells and newly EPI cells along with developmental progress, whereas OCT4 expressed in almost all the cells at the same time. Consistently, analysis of spatiotemporal distribution of SOX2 and the TE marker CDX2 revealed an exclusive expression pattern in D6 blastocysts, whereas no correlation was observed between OCT4 and CDX2 at the same stage. Conclusions: Our results provide a molecular basis in porcine embryonic patterning and a clue for further studying porcine pluripotent regulatory networks. Developmental Dynamics 244:619–627, 2015. © 2015 Wiley Periodicals, Inc.

Fetal gut-like adenocarcinoma is a rare carcinoma, previously reported in the stomach, which features clear cytoplasm cell papillotubular carcinoma and morphology similar to the fetal gut epithelium. SALL4, a novel marker for progenitor-type tumors, has shown high expression in fetal gut-like adenocarcinoma, which may represent oncofetal protein expression and fetal differentiation. Herein, we report the first case of fetal gut-like adenocarcinoma of the duodenum. An 81-year-old man was admitted on account of jaundice. A deeply ulcerating duodenal tumor formed a 4 cm periampullar mass. Pancreatoduodenectomy with lymphadenectomy was performed. Funicular and solid growth of clear cytoplasmic cells that resembled the fetal gut epithelium formed a poorly differentiated adenocarcinoma. Lymph node metastasis was present. Immunohistochemistry analysis showed diffuse nuclear positivity for SALL4. For postoperative therapy, S-1 was administered. The patient is alive with no recurrence, 83 months postoperatively. This case may contribute to further elucidate the disease entity of SALL4 positive, fetal-type carcinomas.

Aims To establish a diagnostic immunohistochemical panel for various histotypes of yolk sac (primitive endodermal) tumours (YSTs) by comparison with the human yolk sac (HYS) immunophenotype. Methods and results Twenty-five YSTs showing either classical patterns (CPs) of histology (microcystic/reticular, n = 14; polyvesicular, n = 1; and hepatoid, n = 1) or somatic glandular patterns (SGPs; n = 9) were analysed for expression of α-fetoprotein (AFP), glypican-3 (GPC3), villin, hepatocyte paraffin-1 (HepPar-1), CDX2, SALL4 and LIN28. AFP expression was constantly heterogeneous in CPs but tended to be focal/absent in SGPs. GPC3 was diffuse in CPs but heterogeneous (seven cases) or focal/absent (two cases) in SGPs. HepPar-1 expression was focal in all but three cases (diffuse in one CP-hepatoid and two SGPs). CDX2 positivity was focal in CPs but heterogeneous (seven cases) or diffuse (two cases) in SGPs. Villin, SALL4 and LIN28 were diffusely positive in nearly all cases. Conclusions CPs reproduce the immunophenotype of HYS and early endoderm with variable expression of both AFP and markers of early gut or hepatic differentiation. SGPs with intestinal differentiation often have incomplete immunophenotypes. A differential diagnosis panel, including both markers of pluripotentiality (SALL4 and/or LIN28) and endoderm (AFP, GPC3 and villin), is proposed. It identifies overlapping multidifferentiation of primitive and somatic immunophenotypes, supporting the recently proposed term of primitive endodermal tumours.

SALL4, as a stemness marker, plays a key role in the maintenance of pluripotency and self-renewal of cancer stem cells. To elucidate probable linkage between SALL4 stemness marker and bone morphogenetic protein (BMP) cell signaling pathway, we aimed to analyze the expression levels of the related genes in esophageal squamous cell carcinoma (ESCC) patients. Tumoral and corresponding margin normal tissues from 50 treatment-naive ESCC patients were subjected for expression analysis using relative comparative real-time reverse transcription polymerase chain reaction. There were significant correlations between SALL4 mRNA and BMP signaling target genes expression including SIZN1, VENTX, and DIDO1 (P < 0.01). Tight associations of gene expression were observed in primary stages of tumor progression (stages I/II), and the invaded tumors to the adventitia (T3/T4). Furthermore, significant correlations between the expression of BMP signaling target genes were observed (P < 0.01). SALL4 may play role in tumorigenesis and tumor cell invasiveness of ESCC through correlation with BMP signaling genes.

In cell cultures, the dispersed phenotype is indicative of the migratory ability. Here we characterized Sal-like 4 (SALL4) as a dispersion factor in basal-like breast cancer. Our shRNA-mediated SALL4 knockdown system and SALL4 overexpression system revealed that SALL4 suppresses the expression of adhesion gene CDH1, and positively regulates the CDH1 suppressor ZEB1. Cell behavior analyses showed that SALL4 suppresses intercellular adhesion and maintains cell motility after cell–cell interaction and cell division, which results in the dispersed phenotype. Our findings indicate that SALL4 functions to suppress CDH1 expression and to maintain cell dispersion in basal-like breast cancer.

Aims SALL4 is a marker of germ cell tumours. The aim of this study was to investigate SALL4 expression in blastemal tumours, particularly in hepatoblastoma. Methods and results The study included 12 hepatoblastomas. Eight hepatoblastomas were pure epithelial tumours, and four were mixed epithelial and mesenchymal tumours. The patients were nine males and three females with a mean age of 14.6 months. Immunohistochemistry was performed with an antibody against SALL4, using an automated immunostainer. Seven of 12 hepatoblastomas showed nuclear staining only in the embryonal component. Fetal and mesenchymal components were negative. Conclusions SALL4 is expressed in blastemal tumours, particularly in the embryonal subtype of hepatoblastoma. Pathologists need to be aware of such expression so that misdiagnosis can be avoided.

Aim: Primary ovarian malignant germ cell tumors (OMGCTs) are rare and difficult to diagnose. Immunohistochemistry can help in the diagnosis and development of new management strategies. The aim of this study was to investigate the frequency of CD117, CD133, SALL4, OCT4, TCL1 and glypican-3 marker expression in OMGCTs. Material and Methods: We examined the expression of six markers in 87 (85 pure and two mixed OMGCTs) cases of OMGCT using immunohistochemical staining. Staining was graded in a semiquantitative manner as follows: negative (no staining), 1+ (1–30% staining), 2+ (31–60% staining), or 3+ (>60% staining). Results: All 27 dysgerminomas and all 31 YSTs showed CD117 expression, with only nine (29%) positively stained in immature teratomas. SALL4 and glypican-3 were strongly positive in 100 and 79.3%, respectively, of YSTs. All dysgerminomas were positive for OCT4, whereas all YSTs and immature teratomas were negative. 100% of dysgerminomas were positive for TCL1, but all immature teratomas were negative. CD133 expression showed generally the same tendency in the 3 OMGCTs. Conclusion: CD117 can be used as a diagnostic marker for dysgerminoma and YST. SALL4 is a more sensitive and specific marker for YSTs than glypican-3. SALL4 and OCT4 are useful in distinguishing YST from dysgerminoma.

We review the current knowledge on human yolk sactumours (YSTs) 50 years after their initial description.Their complex nomenclature and histogenesis stressthe fact that they are not a discrete entity, but representa multifaceted group of neoplasms, for which the termprimitive endodermal tumours would be more appropri-ate, accounting for their capacity to differentiate intovarious extraembryonal and somatic cell types. Differ-ent histological patterns of human YSTs correlate withthe developmental potential of primitive endoderm andmesenchyme, but they are also similar to some murineexperimental tumours. Exceptionally, YSTs replicatethe tubular structures of the human yolk sac andallantois. Endodermal somatic differentiation repro-duces pulmonary, intestinal and hepatic tissues andare identical with some, embryonal-type endodermal,gastric and lung carcinomas, which are indistinguish-able from YSTs. YSTs may show an overgrowth of theirmesenchymal (sarcomatous) and epithelial compo-nents (such as mucinous carcinoma or carcinoid) andalso be a source of haematological malignancies. YSTsassociated with non-germ cell tumours probably orig-inate from malignant pluripotent somatic stem cells.Only AFP and glypican-3 are characteristic immuno-histochemical markers. Pluripotent antibodies (SALL4,Lin28, IMP-3) help in differential diagnoses, whilesome differentiation markers (CDX2, TTF-1, HepPar1)facilitate recognition of unusual variants of YSTs.Keywords: AFP, glypican-3, human yolk sac, nomenclature, primitive endodermal tumours, yolk sac tumourAbbreviations: AFP, alpha-foetoprotein; HepPar1, hepatocyte paraffin 1; TTF-1, thyroid transcription factor 1;YST, yolk sac tumourYolk sac tumours (YSTs) are intriguing neoplasms;possibly few tumours have been known by moreambiguous names in recent history. The concepts thatled to their identification tell a fascinating story, oftenreminiscent of a detective tale with an abundance of redherrings, due to their occurrence in different sites, theirvaried histology and the interpretation of the relation-ships between experimental and human pathology.

Although aneuploidy is commonly observed in human cancers, the molecular mechanism underlying aneuploidization remains unclear. We used multiploid cancer model that had diploid and aneuploid cancer cells within the same cancerous tissue and attempted to detect specific epigenetic alterations associated with tumor cell aneuploidy. Thirty-four multiploid colorectal cancers were subjected to crypt isolation and cell sorting, and paired diploid and aneuploid cancer cells were separated from each cancerous tissue. A methylated CpG island amplification provided a considerable number of CpG sequences that showed different methylation status between the above 2 cell populations. BLAST homology search revealed 24 different candidates (11 hypermethylated and 13 hypomethylated) from these sequences. The putative promoter sequence of the SALL4 (sal-like 4, a human homolog to Drosophila spalt) gene was particularly more frequently hypermethylated in aneuploid cells (62%) than diploid ones (35%) in the 34 multiploid cancers. Moreover, such hypermethylation occurred more often in aneuploid cancers (8 of 16, 50%) than diploid cancers (3 of 18, 17%). In combination with demethylation study on cultured cells, these results implied a possible association between epigenetic silencing of SALL4 and tumor cell aneuploidy. SALL4 may be one of important key players that act as “caretakers” for chromosomal stability. Our new approach is a powerful tool for the global identification of such key players. © 2007 Wiley-Liss, Inc.

The IVIC syndrome described in 1980 in a large Venezuelan family, is an autosomal dominant condition characterized by upper limbs anomalies (radial ray defects, carpal bones fusion), extraocular motor disturbances, congenital bilateral non-progressive mixed hearing loss; other less consistent malformations include heart involvement, mild thrombocytopenia and leukocytosis (before age 50), shoulder girdle hypoplasia, imperforate anus, kidney malrotation or rectovaginal fistula. Since 2002, mutations in the SALL4 locus have been reported producing phenotypic features quite similar to those in IVIC syndrome; this gene was thus proposed as a candidate for the condition. A segregation analysis of four SNPs in exon 2 (c.1520T > G, c.1860A > G, c.2037C > T, and c.2392A > C) was carried out in 14 affected and in 15 normal family members. Haplotype T;A;C;A was found to always segregate with the disease. Sequencing the whole coding regions revealed one heterozygous base deletion in exon 3 (c.2607delA) causing a premature stop signal 44 codons downstream (p.Q869fsX44) which segregates with the phenotype, being absent in controls. The large number of affected individuals presumably carrying the same mutation (n = 26) with quite different degrees of involvement allowed a discussion about possible mechanisms for the SALL4 action. The finding of a SALL4 mutation in a family with such a wide pleiotropic spectrum proves that at least Okihiro, acro-renal-ocular and IVIC syndromes are allelic entities.

The SALL4 gene encodes a putative zinc finger transcription factor and is located on chromosome 20q13.13-13.2. Mutations in SALL4 have been identified in patients with Okihiro syndrome, which is characterized by radial ray anomalies associated with a Duane anomaly. Here, we report an unusual family in which affected persons show an extremely variable phenotype consistent with either Okihiro syndrome, hemifacial microsomia, or isolated Duane anomaly. A novel nonsense mutation in the SALL4 gene was detected in all affected family members and obligate carriers. This mutation is located in exon 3, only 29 bp 5′ of the most 3′ intron, and would therefore be expected to escape the nonsense mediated mRNA decay pathway, which might explain the phenotypic variablity and mild degree of limb involvement. © 2006 Wiley-Liss, Inc.

The multi-C2H2 zinc-finger domain containing transcriptional regulators of the spalt (SAL) family plays important developmental regulatory roles. In a competitive subtractive hybridization screen of genes expressed in Xenopus laevis hindlimb regeneration blastemas, we identified a SAL family member that, by phylogenetic analysis, falls in the same clade as human SALL4 and have designated it as XlSALL4. Mutations of human SALL4 have been linked to Okihiro syndrome, which includes preaxial (anterior) limb defects. The expression pattern of XlSALL4 transcripts during normal forelimb and hindlimb development and during hindlimb regeneration at the regeneration-competent and regeneration-incompetent stages is temporally and regionally dynamic. We show for the first time that a SAL family member (XlSALL4) is expressed at the right place and time to play a role regulating both digit identity along the anterior/posterior axis and epimorphic limb regeneration. Developmental Dynamics 233:356–367, 2005. © 2005 Wiley-Liss, Inc.

A two-generation family is reported in which three members have Duane anomaly and distal limb abnormalities. All three affected have photopic electroretinogram responses that are abnormal or at the lower limit of the normal range with normal scotopic responses. Two affected family members also have hearing loss. The likeliest diagnosis is the syndrome listed as "arthrogryposis-ophthalmoplegia syndrome" on the London Dysmorphology Database or as "arthrogryposis with oculomotor limitation and electroretinal abnormalities" or "oculomelic aplasia" in OMIM [MIM 108145]. In view of the similarities with Okihiro syndrome, a search for mutations within the SALL4 gene was undertaken, but none were identified.