Establishment and characterization of Roberts syndrome and SC phocomelia model medaka (Oryzias latipes)

Roberts syndrome and SC phocomelia (RBS/SC) are genetic autosomal recessive syndromes caused by establishment of cohesion 1 homolog 2 ( ESCO 2) mutation. RBS/SC appear to have a variety of clinical features, even with the same mutation of the ESCO2 gene. Here, we established and genetically characterized a medaka model of RBS/SC by reverse genetics. The RBS/SC model was screened from a mutant medaka library produced by the Targeting Induced Local Lesions in Genomes method. The medaka mutant carrying the homozygous mutation at R80S in the conserved region of ESCO2 exhibited clinical variety (i.e. developmental arrest with craniofacial and chromosomal abnormalities and embryonic lethality) as characterized in RBS/SC. Moreover, widespread apoptosis and downregulation of some gene expression, including notch1a, were detected in the R80S mutant. The R80S mutant is the animal model for RBS/SC and a valuable resource that provides the opportunity to extend knowledge of ESCO2. Downregulation of some gene expression in the R80S mutant is an important clue explaining non-correlation between genotype and phenotype in RBS/SC.     

Inactivating mutations in ESCO2 cause SC phocomelia and Roberts syndrome: no phenotype-genotype correlation

The rare, autosomal recessive Roberts syndrome (RBS) is characterized by tetraphocomelia, profound growth deficiency of prenatal onset, craniofacial anomalies, microcephaly, and mental deficiency. SC phocomelia (SC) has a milder phenotype, with a lesser degree of limb reduction and with survival to adulthood. Since heterochromatin repulsion (HR) is characteristic for both disorders and is not complemented in somatic-cell hybrids, it has been hypothesized that the disorders are allelic. Recently, mutations in ESCO2 (establishment of cohesion 1 homolog 2) on 8p21.1 have been reported in RBS. To determine whether ESCO2 mutations are also responsible for SC, we studied three families with SC and two families in which variable degrees of limb and craniofacial abnormalities, detected by fetal ultrasound, led to pregnancy terminations. All cases were positive for HR. We identified seven novel mutations in exons 3-8 of ESCO2. In two families, affected individuals were homozygous--for a 5-nucleotide deletion in one family and a splice-site mutation in the other. In three nonconsanguineous families, probands were compound heterozygous for a single-nucleotide insertion or deletion, a nonsense mutation, or a splice-site mutation. Abnormal splice products were characterized at the RNA level. Since only protein-truncating mutations were identified, regardless of clinical severity, we conclude that genotype does not predict phenotype. Having established that RBS and SC are caused by mutations in the same gene, we delineated the clinical phenotype of the tetraphocomelia spectrum that is associated with HR and ESCO2 mutations and differentiated it from other types of phocomelia that are negative for HR.     

Chromatid repulsion associated with Roberts/SC phocomelia syndrome is reduced in malignant cells and not expressed in interspecies somatic-cell hybrids.

Different cell types from a female patient with Roberts/SC phocomelia syndrome were evaluated quantitatively for the presence of repulsion of heterochromatin and satellite regions of mitotic chromosomes. Whereas EBV-transformed lymphoblasts from an established cell line revealed these phenomena at frequencies equal to those in PHA-stimulated lymphocytes and cultured skin fibroblasts, aneuploid cells from a metastatic melanoma displayed them at 50% lower frequency. Cocultivation of the patient's fibroblasts with either an immortal Chinese hamster cell line or with a human male fibroblast strain carrying a t(4;6)(p14;q21) translocation showed that the phenomenon was not corrected or induced by a diffusible factor or by cell-to-cell contact. In each experiment, only the patient's metaphase spreads revealed chromatid repulsion. In fusion hybrids between the patient's fibroblasts and an established Chinese hamster cell line, the human chromosomes behaved perfectly normally, suggesting that the gene product which is missing or mutant in Roberts/SC phocomelia syndrome is supplied by the Chinese hamster genome.     

A prospective evaluation of the prevalence of submucous cleft palate in patients with isolated cleft lip versus controls.

Although there is an established relationship between cleft lip and overt cleft palate, the relationship between isolated cleft lip and submucous cleft palate has not been investigated. To test the hypothesis that patients with isolated cleft lip have a greater association with submucous cleft palate, a double-armed prospective trial was designed. A study group of 25 consecutive children presenting with an isolated cleft lip, with or without extension through the alveolus but not involving the secondary palate, was compared with a control group of 25 children with no known facial clefts. Eligible patients were examined for the presence of physical criteria associated with classic submucous cleft palate, namely, (1) bifid uvula, (2) absence of the posterior nasal spine, and (3) zona pellucida. Nasoendoscopy was subsequently performed just after induction of general anesthesia, and the findings were correlated with digital palpation of the palatal muscles. Patients who did not satisfy all three physical criteria and in whom nasoendoscopy was distinctly abnormal relative to the control group were classified as having occult submucous cleft palate. Classic submucous cleft palate was found in three study group patients (12 percent), all of whom had flattening or a midline depression of the posterior palate and musculus uvulae on nasoendoscopy and palpable diastasis of the palatal muscles under general anesthesia. An additional six study group patients (24 percent) had similar nasoendoscopic criteria and palpable diastasis of the palatal muscles; they were classified as having occult submucous cleft palate. No submucous cleft palate was identified in the control group. Seventeen patients in the study group had an alveolar cleft with a 53 percent (9 of 17) prevalence of submucous cleft palate. In the present study, classic submucous cleft palate in association with isolated cleft lip was 150 to 600 times the reported prevalence in the general population. All children with an isolated cleft lip should undergo peroral examination and speech/resonance assessment no later than the age of 3 years. Any child with an isolated cleft lip with velopharyngeal inadequacy or before an adenoidectomy should be assessed by flexible nasal endoscopy to avoid missing an occult submucous cleft palate.     

A locus for isolated cleft palate, located on human chromosome 2q32.

We present evidence for the existence of a novel chromosome 2q32 locus involved in the pathogenesis of isolated cleft palate. We have studied two unrelated patients with strikingly similar clinical features, in whom there are apparently balanced, de novo cytogenetic rearrangements involving the same region of chromosome 2q. Both children have cleft palate, facial dysmorphism, and mild learning disability. Their karyotypes were originally reported as 46, XX, t(2;7)(q33;p21) and 46, XX, t(2;11)(q33;p14). However, our molecular cytogenetic analyses localize both translocation breakpoints to a small region between markers D2S311 and D2S116. This suggests that the true location of these breakpoints is 2q32 rather than 2q33. To obtain independent support for the existence of a cleft-palate locus in 2q32, we performed a detailed statistical analysis for all cases in the human cytogenetics database of nonmosaic, single, contiguous autosomal deletions associated with orofacial clefting. This revealed 2q32 to be one of only three chromosomal regions in which haploinsufficiency is significantly associated with isolated cleft palate. In combination, our data provide strong evidence for the location at 2q32 of a gene that is critical to the development of the secondary palate. The close proximity of these two translocation breakpoints should also allow rapid progress toward the positional cloning of this cleft-palate gene.     

Genetics of cleft lip and cleft palate in China.

During the past 10 years, 60 cases of cleft lip with or without cleft palate [CL(P)] were recorded among 45,072 newborns at Shanghai International Peace Maternity and Infant Hospital, China. The incidence was 1.33 per 1,000 births. The family histories of 163 CL(P) patients were analyzed. The incidences of CL(P) in the first-, second-, and third-degree relatives of CL(P) patients were 11/246 (4.47%), 10/1,032 (0.97%), and 6/1,727 (0.35%), respectively. Of the 163 probands, three had a history of consanguinity of the parents (1.8%), in contrast to 0.77% in the general population. These data are suggestive of multifactorial inheritance. The heritability of CL(P) in our study calculated by Falconer's formula was 77.6%.     

Familial recurrence-pattern analysis of cleft lip with or without cleft palate.

Cleft lip with or without cleft palate (CL/P) is a common congenital malformation with an incidence in European white populations of about 1/1,000. The familial clustering of CL/P has been extensively characterized, and epidemiological studies have proposed monogenic models (with reduced penetrance), multifactorial/threshold models, and mixed major-gene/multifactorial models to explain its inheritance. The recognition of an association between two RFLPs at the transforming growth factor alpha (TGFA) locus and CL/P supports a major-gene component to the etiology of CL/P. Risch has shown that the recurrence risk ratio lambda R (risk to relatives, vs. population prevalence) is a useful pointer to the mode of inheritance. Here we further develop the use of lambda R to analyze recurrence-risk data for CL/P. Recurrence risks for first-, second-, and third-degree relatives equate well with oligogenic models with as few as four loci. A monogenic/additive model is strongly rejected. The limited available twin data are also consistent with this model. A "major gene" interacting epistatically with an oligogenic background is shown to be a plausible alternative. Power calculations for a linkage study to map the CL/P major-risk locus suggest that a sample of 50 affected sib pairs will be adequate, but linkage to minor-risk loci will require very much larger samples.     

Segregation analysis of cleft lip with or without cleft palate in the First Nations (Amerindian) people of British Columbia and review of isolated cleft palate etiologies

BACKGROUND: The First Nations (Amerindian) population of British Columbia, Canada, has the highest reported birth prevalence in the world of cleft lip with or without cleft palate (CL/P) at nearly 3 per 1000 births. In addition, a substantial proportion of cleft palate only (CPO) cases in this population has been reported to be X‐linked. The aims of this study were to perform complex segregation analysis to investigate the mode of inheritance of CL/P in the First Nations people of British Columbia and to review the etiology of the CPO cases. METHODS: All First Nations children born in British Columbia between 1952 and 1971 with an orofacial cleft were included in the study. Multiple sources of ascertainment were used, so that nearly 100% of live births were identified and included during this time. No stillbirths were found but would likely have been ascertained. Extended pedigrees were constructed from these probands and examination of immediate family members, e.g., parents and siblings, was done wherever possible. Complex segregation analysis included all family members. In addition, a CPO case review was conducted. RESULTS: Complex segregation analysis supports the hypothesis that the most likely mode of inheritance of CL/P in this population is a mixed model; that is, an autosomal major gene with polygenic component. The review of 26 CPO cases showed that a substantial proportion are syndromic. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Distichiasis and cleft palate

A patient with the unusual coexistence of distichiasis and cleft palate is described. Distichiasis is a rare congenital eyelid anomaly in which accessory eyelashes are present in the meibomian gland orifices. Its association with other systemic abnormalities is reviewed; the distinctions among distichiasis, trichiasis, entropion, and epiblepharon are outlined; and methods of treatment for distichiasis are described.     

Cleft lip, cleft palate, and velopharyngeal insufficiency

This article provides an introduction to the anatomical and clinical features of the primary deformities associated with unilateral cleft lip-cleft palate, bilateral cleft lip-cleft palate, and cleft palate. The diagnosis and management of secondary velopharyngeal insufficiency are discussed. The accompanying videos demonstrate the features of the cleft lip nasal deformities and reliable surgical techniques for unilateral cleft lip repair, bilateral cleft lip repair, and radical intravelar veloplasty.