Evidence of a locus for orofacial clefting on human chromosome 6p24 and STS content map of the region

Orofacial clefting is genetically complex, no single gene beingresponsible for all forms. It can, however, result from a singlegene defect either as part of a syndrome (e.g. van der Woudesyndrome, Treacher—Colllns syndrome, velo–cardio–facialsyndrome) or as an Isolated phenotypic effect (e.g. X–linkedcleft palate; non–syndromlc, autosomal dominant orofacialclefting). Several studies have suggested that chromosome 6pis a candidate region for a locus involved in orofacial clefting.We have used YAC clones from contigs in 6p25–p23 to investigatethree unrelated cases of cleft lip and palate coincident withchromosome 6p abnormalities. Case 1 has bilateral cleft lipand palate and a balanced translocation reported as 46, XY,t(6, 7)(p23; q36.1). Case 2 has multiple abnormalities Includingcleft lip and palate and was reported as 46, XX, del(6)(p23;pter). Case 3 has bilateral cleft lip and palate and carriesa balanced translocation reported as 46, XX, t(6; 9)(p23;q22.3).We have Identified two YAC clones, both of which cross the breakpointin cases 1 and 3 and are deleted in case 2. These clones mapto 6p24.3 and therefore suggest the presence of a locus fororofacial clefting in this region. The HGP22 and AP2 genes,potentially involved in face formation, have been found to flankthis region, while F13A maps further telomeric in 6p24.3/25.     

Genetic variation in the proto-oncogene SKI and risk for orofacial clefting

BACKGROUND: SKI is a proto-oncogene that is required for development of the central nervous system and skeletal muscle, and is involved in specifying selected cranial neural-crest-derived craniofacial structures. To identify genetic variants within the SKI gene and investigate the potential association between SKI polymorphisms and risk for orofacial defects, we initially re-sequenced the gene. METHODS: DNA re-sequencing of all seven exons of the SKI gene was performed on 100 control samples. Subsequently, we genotyped 394 samples (148 CLP cases, 99 CP cases, and 147 control infants) for a novel SNP identified in the DNA re-sequencing effort using restriction fragment length polymorphism (RFLP) analysis. RESULTS: We identified one polymorphism in exon 1 of the SKI gene (257C>G) from controls. This SNP resulted in an amino acid change from alanine to glycine (A62G, GenBank Accession No. NM_003036). Among all samples genotyped by the RFLP method, variants (CG, GG) were found in 10.5% of the cases, compared to a prevalence of 17.7% in the controls. The odds ratio was calculated to be 0.6, with a 95% confidence interval (CI) of 0.3-1.0. CONCLUSION: In a population of California infants with craniofacial defects, a novel polymorphism of the SKI gene was found to be associated with a decreased risk for orofacial defects. The function of this polymorphism and how it might confer protection to the embryo against craniofacial malformations is currently under investigation in our laboratory.     

Alveolar soft-part sarcoma: Further evidence by FISH for the involvement of chromosome band 17q25

A cytogenetic study of an alveolar soft-part sarcoma, a rare tumor of probably myogenic origin, demonstrated a t(X;17)(p11;q25) as the sole chromosomal abnormality. Dual- and triple-color fluorescence in situ hybridization, performed on metaphase and interphase cells, confirmed the translocation between chromosomes X and 17 and demonstrated that this translocation resulted in loss of 17q25. Involvement of 17q25 has been described in four previously published cases of alveolar soft-part sarcoma, but without further characterization. Compared to our karyotype, it seems that the derivative chromosome 17 observed in the reported cases could also be the result of a t(X;17) with possible loss of the 17q25 band. If so, a 17q25 deletion and/or chromosome rearrangement between Xp and 17q leading either to a gene fusion or gene disruption could play an important role in the pathogenesis of alveolar soft-part sarcoma. Genes Chromosomes Cancer 23:194–197, 1998. © 1998 Wiley-Liss, Inc.     

Further evidence for a relationship between the 5p15 chromosome region and the oculoauriculovertebral anomaly

The oculoauriculovertebral anomaly (OAV) or Goldenhar syndrome is a malformation complex that has been described in several chromosomal rearrangements. Among them a deletion of the terminal 5p has recurred in seven previous patients. We wish to report on an additional such patient in order to reinforce the significance of this genomic region in the cause of at least a subgroup of OAV cases. Future studies, particularly in the OAV patients with a lateral facial cleft, might define one genetic background of the disorder. Our patient had a complex translocation chromosome 45,XX, inv(2) (q32q37)mat, dic(5;21) (p15.3;q22.3)dn, resulting in a terminal 5p deletion, a terminal deletion of 21q demonstrated by FISH studies, and a duplication of 21q22.11-q22.12 documented by molecular karyotyping. In addition to OAV she developed myelodysplasia treated with bone marrow transplantation. We discuss her clinical findings with reference to her karyotype findings and review the patients with OAV and a terminal deletion of 5p.     

Mapping of RXRB to human chromosome 6p21. 3

Retinoid X Receptor beta (RXRB) is a member of the retinoid X receptor (RXR) family of nuclear receptors which are involved in mediating the effects of retinoic acid (RA). We have confirmed the localization of RXRB to chromosome 6 and we have mapped the gene to chromosome 6p21. 3-p21.1 by PCR amplification of 5′ untranslated sequence in panels of rodent-human somatic cell hybrids and to 6p21.3 by fluorescent in situ hybridization.     

Three-dimensional morphometric analysis of brain shape in nonsyndromic orofacial clefting

Previous studies report structural brain differences in individuals with nonsyndromic orofacial clefts (NSOFC) compared with healthy controls. These changes involve non‐uniform shifts in tissue volume within the cerebral cortex and cerebellum, suggesting that the shape of the brain may be altered in cleft‐affected individuals. To test this hypothesis, a landmark‐based morphometric approach was utilized to quantify and compare brain shape in a sample of 31 adult males with cleft lip with or without cleft palate (CL/P), 14 adult males with cleft palate only (CPO) and 41 matched healthy controls. Fifteen midline and surface landmarks were collected from MRI brain scans and the resulting 3D coordinates were subjected to statistical shape analysis. First, a geometric morphometric analysis was performed in three steps: Procrustes superimposition of raw landmark coordinates, omnibus testing for group difference in shape, followed by canonical variates analysis (CVA) of shape coordinates. Secondly, Euclidean distance matrix analysis (EDMA) was carried out on scaled inter‐landmark distances to identify localized shape differences throughout the brain. The geometric morphometric analysis revealed significant differences in brain shape among all three groups (P < 0.001). From CVA, the major brain shape changes associated with clefting included selective enlargement of the anterior cerebrum coupled with a relative reduction in posterior and/or inferior cerebral portions, changes in the medio‐lateral position of the cerebral poles, posterior displacement of the corpus callosum, and reorientation of the cerebellum. EDMA revealed largely similar brain shape changes. Thus, compared with controls, major brain shape differences were present in adult males with CL/P and CPO. These results both confirm and expand previous findings from traditional volumetric studies of the brain in clefting and provide further evidence that the neuroanatomical phenotype in individuals with NSOFC is a primary manifestation of the defect and not a secondarily acquired characteristic.     

Linkage of eye movement dysfunction to chromosome 6p in schizophrenia: additional evidence.

Establishing the genetics of physiological traits associated with schizophrenia may be an important first step in building a neurobiological bridge between the disease phenotype and its genetic underpinnings. One of the best known of the traits associated with schizophrenia is a disorder of smooth pursuit eye tracking (ETD), which is present in 50-80% of schizophrenia patients. ETD is more than three times more prevalent in the families of a schizophrenia patient than is schizophrenia itself. Arolt et al. [1999] estimated LOD scores for ETD of 2.85 for D6S282 and 3.70 for D6S271, two markers on 6p21.1, as well as obtaining an indication of possible linkage for schizophrenia. Our sample comprised two large families in Denmark. Markers in the region that was implicated by the study of Arolt et al. [1996, 1999] were analyzed as part of a genome scan using the "latent trait (L.T.) model" for the co-transmission of schizophrenia and ETD that we had previously fitted to segregation analysis data from Norway. We obtained a LOD score of 2.05 for D6S1017, a marker within 3 cM of the positive markers obtained by Arolt et al. [1996, 1999]. We regard our results as independent evidence supporting the findings of Arolt et al. [1996, 1999] and also as support for the L.T. model as a way of combining the traits ETD and schizophrenia.     

Further evidence for adrenergic transmission in the human vas deferens.

1. Isolated portions of human vas deferens responded to field stimulation of the intramural nerve fibres or to exogenously applied noradrenaline with rhythmical contractions of both longitudinal and circular muscle layers. 2. In guinea-pig and rabbit vas deferens field stimulation produced an initial rapid 'twitch' response which was not found with human vasa. 3. Responses of the human vas to field stimulation were depressed by the alpha-adrenoceptor blocking agents phentolamine and yohimbine. 4. It is concluded that the motor innervation of the human vas deferens is adrenergic and the relevance of this to the physiological operation of the tissue is discussed.     

IRF6 mutation screening in non‐syndromic orofacial clefting: analysis of 1521 families

Van der Woude syndrome (VWS) is an autosomal dominant malformation syndrome characterized by orofacial clefting (OFC) and lower lip pits. The clinical presentation of VWS is variable and can present as an isolated OFC, making it difficult to distinguish VWS cases from individuals with non‐syndromic OFCs. About 70% of causal VWS mutations occur in IRF6, a gene that is also associated with non‐syndromic OFCs. Screening for IRF6 mutations in apparently non‐syndromic cases has been performed in several modestly sized cohorts with mixed results. In this study, we screened 1521 trios with presumed non‐syndromic OFCs to determine the frequency of causal IRF6 mutations. We identified seven likely causal IRF6 mutations, although a posteriori review identified two misdiagnosed VWS families based on the presence of lip pits. We found no evidence for association between rare IRF6 polymorphisms and non‐syndromic OFCs. We combined our results with other similar studies (totaling 2472 families) and conclude that causal IRF6 mutations are found in 0.24–0.44% of apparently non‐syndromic OFC families. We suggest that clinical mutation screening for IRF6 be considered for certain family patterns such as families with mixed types of OFCs and/or autosomal dominant transmission.     

Further evidence for linkage of low-mid frequency hearing impairment to the candidate region on chromosome 4p16.3

We have investigated a three-generation family with an autosomal dominant low-mid frequency hearing loss. Audiograms show consistently a hearing threshold of 50+/-20 db hearing loss (HL) between 250 Hz and 1-2 kHz. Normal hearing level was reached between 3 and 6 kHz in all examined children. Adult patients show an additional hearing impairment (HI) in the mid and higher frequencies that seems to differ from presbyacusis. The HI is always bilateral and symmetrical. Genes causing non-syndromic autosomal-dominant deafness with HI in the low and mid frequencies were previously mapped to chromosome 4p16.3 (DFNA6, DFNA14) and chromosome 5q31 (DFNA1). After exclusion of linkage to DFNA1 on chromosome 5, we mapped the candidate gene region to the DFNA14 and DFNA6 loci, between the genetic markers D4S432 and D4S431, located on chromosome 4. This is a further family in which evident linkage of low-mid frequency HI to the candidate region on chromosome 4p16.3 has been found.     

Further evidence for an imprinted gene for neonatal diabetes localised to chromosome 6q22-q23

Transient neonatal diabetes mellitus (TNDM) is a rare form of childhood diabetes which usually resolves in the first 6 months of life but which predisposes to type 2 diabetes of adult onset. We recently reported paternal uniparental isodisomy of chromosome 6 (UPD6) in two children with TNDM and proposed that there may be an imprinted gene important in the aetiology of diabetes on chromosome 6. We now describe two unrelated families which independently suggest that the gene is imprinted, is paternally expressed and maps to 6q22-q23. One family has a duplication while the other, with familial TNDM, shows linkage to a marker in this region.      

Prediction of liability to orofacial clefting using genetic and craniofacial data from parents.

BACKGROUND: Cleft lip with or without cleft palate (CL(P)) and isolated cleft palate (CP) are separate clinical entities and for both polygenic multifactorial aetiology has been proposed. Parents of children with orofacial clefting have been shown to have distinctive differences in their facial shape when compared to matched controls. OBJECTIVE: To test the hypothesis that genetic and morphometric factors predispose to orofacial clefting and that these markers differ for CL(P) and CP. Methods-Polymorphisms at the transforming growth factor alpha (TGFalpha) locus in 83 parents of children with nonsyndromic orofacial clefts were analysed, and their craniofacial morphology was assessed using lateral cephalometry. RESULTS: Parents of children with CL(P) and CP showed an increased frequency of the TGFalpha/TaqI C2 allele (RR=4.10, p=0.009) relative to the comparison group. Also the TGFalpha/BamHI A1 allele was more prevalent in the CP parents. MULTIVARIATE STATISTICAL ANALYSIS: Using stepwise logistic regression analysis the TGFalpha/TaqI C2 polymorphism provides the best model for liability to orofacial clefting. To determine the type of clefting a model involving interaction between the parental TGFalpha/BamHI and TGFalpha/RsaI genotypes showed the best fit. Using genotype only to predict the clefting defect in the children according to parental genotype, 68.3% could be correctly classified. By adding information on craniofacial measurements in the parents, 76% of CP and 94% of CL(P) parents could be correctly classified. CONCLUSIONS: This study provides a model for prediction of liability to orofacial clefting. These findings suggest that different molecular aberrations at the TGFalpha locus may modify the risk for CP and CL(P).     

Molecular cloning and characterization of OSR1 on human chromosome 2p24

During Drosophila hindgut development, bowl, caudal/CDX, brachyenteron/Brachyury/TBX, fork head/FOX, drumstick, lines, and wingless/WNT play important roles. Drosophila bowl gene is homologous to Drosophila odd-skipped (odd) gene and odd-skipped related gene (sob). Here, human OSR1, related to Drosophila odd, was isolated using bioinformatics and cDNA-PCR. OSR1 was found to encode 266 amino-acid protein with three C2H2-type zinc fingers, a tyrosine phosphorylation site (Tyr 203), and several putative PXXP SH3 binding motifs. Three zinc fingers and a tyrosine phosphorylation site were conserved among human OSR1, OSR2, Drosophila odd, sob, and bowl. OSR1 showed 63.6% total amino-acid identity with OSR2. OSR1 gene consisting of three exons was located on human chromosome 2p24. OSR1 mRNA of 2.3-kb in size was detected in adult colon, small intestine, prostate, testis, and fetal lung. OSR1 mRNA was significantly up-regulated in a pancreatic cancer cell line MIA PaCa-2, and was weakly expressed in gastric cancer cell lines OKAJIMA, MKN45, pancreatic cancer cell lines PANC-1, BxPC-3, AsPC-1, PSN-1, Hs766T, and esophageal cancer cell line TE10. Among 10 cases of primary gastric cancer, OSR1 mRNA was up-regulated in 5 cases, and was down-regulated in 2 cases. This is the first report on molecular cloning and characterization of human OSR1.     

Stress induced Straub tail elevation. Further behavioral evidence in rats for the involvement of endorphins in stress

Adult male Sprague-Dawley rats briefly immersed in cold water and forced to swim showed Straub tail elevation, a typical sign of opiate stimulation, upon removal. The presence of Straub tail was a function of degree of immersion and was reversed by naloxone. This suggests the Straub tail response may be a novel behavioral index of stress-induced endorphin release.     

Further evidence for the involvement of microtubules in the intra-axonal movement of noradrenaline storage granules

1. Constricted cat hypogastric nerve/inferior mesenteric ganglion preparations maintained in vitro for 48 hr have been used to study the effects of different concentrations of colchicine on axonal microtubules and on the proximo-distal movement of catecholamine containing dense-cored vesicles in non-myelinated axons.2. In low concentrations (0.03-0.3 mug/ml.), colchicine had no effect on the number of microtubules per axon and did not diminish the amount of noradrenaline accumulating proximal to the constriction.3. Higher concentrations of colchicine (1.0-10 mug/ml.) produced a dramatic reduction in the number of microtubules per axon. This was associated with marked reductions in the number of dense-cored vesicles and the amount of noradrenaline accumulating above the constriction.4. There was some morphological evidence for a structural relationship between dense-cored vesicles and microtubules.5. These results further support the view that axonal microtubules are involved in the relatively fast proximo-distal transport of noradrenaline containing dense-cored vesicles in non-myelinated sympathetic axons.     

Localization of a gene for oculodentodigital syndrome to human chromosome 6q22-q24

Oculodentodigital syndrome (ODD) is a congenital, autosomal dominant disorder which affects the development of the face, eyes, limbs and dentition. Spastic paraparesis is thought to be an occasional manifestation of the disorder. Type III syndactyly, which occurs as part of ODD, has also been reported to occur as an isolated entity. In the current investigation, a total genome search for the location of the ODD locus was instigated and linkage to polymorphic markers located on chromosome 6q established (pairwise Zmax = 9.37; theta = 0.001). Analysis of a large family with type III syndactyly, but atypical facial features, further suggested that isolated type III syndactyly is also located in this same region of the genome.      

The involvement of 6p in melanoma

Karyotype analysis of first passage cells from a melanoma, occurring in a patient with a giant congenital nevus, revealed monosomy of chromosomes #6 and #11, and trisomy of chromosomes #8 and #22. Five marker chromosomes were present, including two ring chromosomes. The origin of three of the marker chromosomes was determined, and all were found to contain the region 6p21.1–6p23. The ring chromosomes were also thought to contain this region, to which the major histocompatibility locus has been mapped. The significance of these findings is discussed in the light of recent reports of chromosome studies of melanoma cells.