Pierre Robin sequence (PRS) is an important subgroup of cleft palate. We report several lines of evidence for the existence of a 17q24 locus underlying PRS, including linkage analysis results, a clustering of translocation breakpoints 1.06-1.23 Mb upstream of SOX9, and microdeletions both approximately 1.5 Mb centromeric and approximately 1.5 Mb telomeric of SOX9. We have also identified a heterozygous point mutation in an evolutionarily conserved region of DNA with in vitro and in vivo features of a developmental enhancer. This enhancer is centromeric to the breakpoint cluster and maps within one of the microdeletion regions. The mutation abrogates the in vitro enhancer function and alters binding of the transcription factor MSX1 as compared to the wild-type sequence. In the developing mouse mandible, the 3-Mb region bounded by the microdeletions shows a regionally specific chromatin decompaction in cells expressing Sox9. Some cases of PRS may thus result from developmental misexpression of SOX9 due to disruption of very-long-range cis-regulatory elements.

We report the identification of six patients with 3q29 microdeletion syndrome. The clinical phenotype is variable despite an almost identical deletion size. The phenotype includes mild-to-moderate mental retardation, with only slightly dysmorphic facial features that are similar in most patients: a long and narrow face, short philtrum, and high nasal bridge. Autism, gait ataxia, chest-wall deformity, and long and tapering fingers were noted in at least two of six patients. Additional features--including microcephaly, cleft lip and palate, horseshoe kidney and hypospadias, ligamentous laxity, recurrent middle ear infections, and abnormal pigmentation--were observed, but each feature was only found once, in a single patient. The microdeletion is approximately 1.5 Mb in length, with molecular boundaries mapping within the same or adjacent bacterial artificial chromosome (BAC) clones at either end of the deletion in all patients. The deletion encompasses 22 genes, including PAK2 and DLG1, which are autosomal homologues of two known X-linked mental retardation genes, PAK3 and DLG3. The presence of two nearly identical low-copy repeat sequences in BAC clones on each side of the deletion breakpoint suggests that nonallelic homologous recombination is the likely mechanism of disease causation in this syndrome.

We present transcriptome analyses of primary cultures of human fetal cells from pregnancies affected with trisomy 21 (t21) and trisomy 13 (t13). Pooled mRNA samples from t21 and t13 cases were used for comparative hybridizations to cDNA arrays with pooled mRNA from normal cells. When the array cDNAs were grouped by chromosomal location the relevant trisomic chromosome could be clearly identified as showing the most significant misregulation. The average level of transcription on the trisomic chromosome was increased only approximately 1.1-fold compared to normal cells on array analysis. Since the karyotype could be accurately predicted by the transcriptome this could provide a novel method of detecting aneusomy of unknown position. Subsequent analysis of individuals cases demonstrated that variation in transcriptional profiles between samples within each class made transcriptional karyotyping difficult without pooling or the use of arrays with a higher proportion of all human cDNAs. Interestingly, consistent differences in the relative expression levels between chromosomes were detected suggesting that genomic control mechanisms may act over larger distances than previously thought. Most (>95%) >+/-2 SD misregulated genes did not map to the trisomic chromosome and significant misregulation was more common in t13 than t21. These data support a model of a subtle primary upregulation of genes on the trisomic chromosome resulting in a secondary, generalized and more extreme transcriptional misregulation. It seems likely that the degree of this misregulation determines the severity of the phenotype in most aneuploidy.

A high level of cytogenetic expression of the rare folate-sensitive fragile site FRA12A is significantly associated with mental retardation. Here, we identify an elongated polymorphic CGG repeat as the molecular basis of FRA12A. This repeat is in the 5' untranslated region of the gene DIP2B, which encodes a protein with a DMAP1-binding domain, which suggests a role in DNA methylation machinery. DIP2B mRNA levels were halved in two subjects with FRA12A with mental retardation in whom the repeat expansion was methylated. In two individuals without mental retardation but with an expanded and methylated repeat, DIP2B expression was reduced to approximately two-thirds of the values observed in controls. Interestingly, a carrier of an unmethylated CGG-repeat expansion showed increased levels of DIP2B mRNA, which suggests that the repeat elongation increases gene expression, as previously described for the fragile X-associated tremor/ataxia syndrome. These data suggest that deficiency of DIP2B, a brain-expressed gene, may mediate the neurocognitive problems associated with FRA12A.

Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1tm1a) that reduces mRNA to ∼10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1tm1a/tm1a). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1tm1a/tm1a embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.

We report fluorescence in situ hybridization (FISH) mapping of 152, mostly de novo, apparently balanced chromosomal rearrangement (ABCR) breakpoints in 76 individuals, 30 of whom had no obvious phenotypic abnormality (control group) and 46 of whom had an associated disease (case group). The aim of this study was to identify breakpoint characteristics that could discriminate between these groups and which might be of predictive value in de novo ABCR (DN-ABCR) cases detected antenatally. We found no difference in the proportion of breakpoints that interrupted a gene, although in three cases, direct interruption or deletion of known autosomal-dominant or X-linked recessive Mendelian disease genes was diagnostic. The only significant predictor of phenotypic abnormality in the group as a whole was the localization of one or both breakpoints to an R-positive (G-negative) band with estimated predictive values of 0.69 (95% CL 0.54-0.81) and 0.90 (95% CL 0.60-0.98), respectively. R-positive bands are known to contain more genes and have a higher guanine-cytosine (GC) content than do G-positive (R-negative) bands; however, whether a gene was interrupted by the breakpoint or the GC content in the 200 kB around the breakpoint had no discriminant ability. Our results suggest that the large-scale genomic context of the breakpoint has prognostic utility and that the pathological mechanism of mapping to an R-band cannot be accounted for by direct gene inactivation.

No abstract

Interstitial deletions of the middle portion of the long arm of chromosome 5 are relatively rare. So far, only 36 cases have been reported. Because of the repetitive banding pattern of this region, the extent and localization of the deleted segment has not been well characterized in the majority of reported cases. This has complicated attempts to establish a definite karyotype-phenotype correlation. We report a further case with a de novo interstitial deletion of the region 5q?15 to 5q?22 identified by standard karyotype analysis. The proband presented with failure to thrive, developmental delay, distinct craniofacial dysmorphic features, and associated structural anomalies (amongst them cleft palate, iris colobomata, and horseshoe kidney, which have previously been reported in 5q deletion cases). In addition, this child had an Arnold-Chiari type I malformation that required surgical decompression. FISH studies using BAC clones spanning the 5q15 to 5q22 region revealed that these were all present in both homologues. Use of more distal clones allowed delineation of the deleted region to 5q22.3q23.3 and to narrow down the breakpoints to approximately 200 kb. The 14 Mb deleted region contains about 60 genes but, with the possible exception of FBN2 and DMXL1, there are no obvious candidate genes for the specific components of the phenotype. This case illustrates the discrepancy between cytogenetic and molecular techniques in trying to delineate 5q interstitial deletions. Molecular studies need to be performed on these patients, to establish genotype-phenotype correlation and to understand the role and influence of genes in this region.