Fibroblast growth factor receptor 3 (FGFR3) transmembrane mutation in Crouzon syndrome with acanthosis nigricans.

Crouzon syndrome, an autosomal dominant condition characterized by craniosynostosis, ocular proptosis and midface hypoplasia, is associated with mutations in fibroblast growth factor receptor 2 (FGFR2) (refs 1-3). For example, we have identified 10 different mutations in the FGFR2 extracellular immunoglobulin III (IgIII) domain in 50% (16/32) of our Crouzon syndrome patients. All mutations described so far for other craniosynostotic syndromes with associated limb anomalies--Jackson-Weiss, Pfeiffer, and Apert--also occur in the extracellular domain of FGFR2, as well as FGFR1 for Pfeiffer syndrome. In contrast, only FGFR3 mutations have been reported in dwarfing conditions--achondroplasia, thanatophoric dysplasia, and hypochondroplasia. For achondroplasia, greater than 99% of mutations occur in the FGFR3 transmembrane domain. We now report the unexpected observation of a FGFR3 transmembrane domain mutation, Ala391Glu, in three unrelated families with Crouzon syndrome and acanthosis nigricans, a specific skin disorder of hyperkeratosis and hyperpigmentation. The association of non-dwarfing and even non-skeletal conditions with FGFR3 mutations reveals the potential for a wide range of FGFR pleiotropic effects as well as locus heterogeneity in Crouzon syndrome. Our study underscores the biologic complexity of the FGFR gene family.

Mutations in TWIST, a basic helix-loop-helix transcription factor, in Saethre-Chotzen syndrome.

Saethre-Chotzen syndrome is one of the most common autosomal dominant disorders of craniosynostosis in humans and is characterized by craniofacial and limb anomalies. The locus for Saethre-Chotzen syndrome maps to chromosome 7p21-p22. We have evaluated TWIST, a basic helix-loop-helix transcription factor, as a candidate gene for this condition because its expression pattern and mutant phenotypes in Drosophila and mouse are consistent with the Saethre-Chotzen phenotype. We mapped TWIST to human chromosome 7p21-p22 and mutational analysis reveals nonsense, missense, insertion and deletion mutations in patients. These mutations occur within the basic DNA binding, helix I and loop domains, or result in premature termination of the protein. Studies in Drosophila indicate that twist may affect the transcription of fibroblast growth factor receptors (FGFRs), another gene family implicated in human craniosynostosis. The emerging cascade of molecular components involved in craniofacial and limb development now includes TWIST, which may function as an upstream regulator of FGFRs.

Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2.

Jackson-Weiss syndrome is an autosomal dominant condition characterized by craniosynostosis, foot anomalies and great phenotypic variability. Recently mutations in fibroblast growth factor receptor 2 (FGFR2) have been found in patients with another craniosynostotic syndrome, Crouzon syndrome. FGFR2 is a member of the tyrosine kinase receptor superfamily, having a high affinity for peptides that signal the transduction pathways for mitogenesis, cellular differentiation and embryogenesis. We now report an FGFR2 mutation in the conserved region of the immunoglobulin IIIc domain in the Jackson-Weiss syndrome family in which the syndrome was originally described. In addition, in four of 12 Crouzon syndrome cases, we identified two new mutations and found two previously described mutations in the same region.

Fibroblast growth factor receptor 2 mutations in Beare-Stevenson cutis gyrata syndrome.

Beare-Stevenson cutis gyrata syndrome (MIM 123790) is an autosomal dominant condition characterized by the furrowed skin disorder of cutis gyrata, acanthosis nigricans, craniosynostosis, craniofacial dysmorphism, digital anomalies, umbilical and anogenital abnormalities and early death. Many of these features are characteristic of some of the autosomal dominant craniosynostotic syndromes. Mutations in Crouzon, Jackson-Weiss, Pfeiffer and Apert syndromes have been reported in the FGFR2 extracellular domain. In Crouzon syndrome patients with acanthosis nigricans, a recurrent mutation occurs in the transmembrane domain of FGFR3. We now describe the detection of FGFR2 mutations in the Beare-Stevenson cutis gyrata syndrome. In three sporatic cases, a novel missense mutation was found causing an amino acid to be replaced by a cysteine; two had the identical Ty375Cys mutation in the transmembrane domain and one had a Ser372Cys mutation in the carboxyl-terminal end of the linker region between the immunoglobulin III-like (Iglll) and transmembrane domains. In two patients, neither of these mutations were found suggesting further genetic heterogeneity.

Revisiting the craniosynostosis-radial ray hypoplasia association: Baller-Gerold syndrome caused by mutations in the RECQL4 gene.

Baller-Gerold syndrome (BGS) is a rare autosomal recessive condition with radial aplasia/hypoplasia and craniosynostosis (OMIM 218600). Of >20 cases reported so far, a few appear atypical and have been reassigned to other nosologic entities, including Fanconi anaemia, Roberts SC phocomelia, and Pfeiffer syndromes after demonstration of corresponding cytogenetic or molecular abnormalities. Clinical overlap between BGS, Rothmund-Thomson syndrome (RTS), and RAPADILINO syndrome is noticeable. Because patients with RAPADILINO syndrome and a subset of patients with RTS have RECQL4 mutations, we reassessed two previously reported BGS families and found causal mutations in RECQL4 in both. In the first family, four affected offspring had craniosynostosis and radial defect and one of them developed poikiloderma. In this family, compound heterozygosity for a R1021W missense mutation and a g.2886delT frameshift mutation of exon 9 was found. In the second family, the affected male had craniosynostosis, radial ray defect, poikiloderma, and short stature. He had a homozygous splice site mutation (IVS17-2A>C). In both families, the affected offspring had craniosynostosis, radial defects, and growth retardation, and two developed poikiloderma. Our results confirm that BGS in a subgroup of patients is due to RECQL4 mutations and could be integrated into a clinical spectrum that encompasses RTS and RAPADILINO syndrome.

High throughput SNP and expression analyses of candidate genes for non-syndromic oral clefts.

BACKGROUND: Recent work suggests that multiple genes and several environmental risk factors influence risk for non-syndromic oral clefts, one of the most common birth defects in humans. Advances in high-throughput genotyping technology now make it possible to test multiple markers in many candidate genes simultaneously. METHODS: We present findings from family based association tests of single nucleotide polymorphism (SNP) markers in 64 candidate genes genotyped using the BeadArray approach in 58 case-parent trios from Maryland (USA) to illustrate how multiple markers in multiple genes can be analysed. To assess whether these genes were expressed in human craniofacial structures relevant to palate and lip development, we also analysed data from the Craniofacial and Oral Gene Expression Network (COGENE) consortium, and searched public databases for expression profiles of these genes. RESULTS: Thirteen candidate genes showed significant evidence of linkage in the presence of disequilibrium, and ten of these were found to be expressed in relevant embryonic tissues: SP100, MLPH, HDAC4, LEF1, C6orf105, CD44, ALX4, ZNF202, CRHR1, and MAPT. Three other genes showing statistical evidence (ADH1C, SCN3B, and IMP5) were not expressed in the embryonic tissues examined here. CONCLUSIONS: This approach demonstrates how statistical evidence on large numbers of SNP markers typed in case-parent trios can be combined with expression data to identify candidate genes for complex disorders. Many of the genes reported here have not been previously studied as candidates for oral clefts and warrant further investigation.

Cleft lip/palate and CDH1/E-cadherin mutations in families with hereditary diffuse gastric cancer.

We report the association of CDH1/E-cadherin mutations with cleft lip, with or without cleft palate (CLP), in two families with hereditary diffuse gastric cancer (HDGC). In each family, the CDH1 mutation was a splicing mutation generating aberrant transcripts with an in-frame deletion, removing the extracellular cadherin repeat domains involved in cell-cell adhesion. Such transcripts might encode mutant proteins with trans-dominant negative effects. We found that CDH1 is highly expressed at 4 and 5 weeks in the frontonasal prominence, and at 6 weeks in the lateral and medial nasal prominences of human embryos, and is therefore expressed during the critical stages of lip and palate development. These findings suggest that alteration of the E-cadherin pathway can contribute to human clefting.

Characterization of the nucleolar gene product, treacle, in Treacher Collins syndrome.

Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development caused by mutations in the gene TCOF1. Its gene product, treacle, consists mainly of a central repeat domain, which shows it to be structurally related to the nucleolar phosphoprotein Nopp140. Treacle remains mostly uncharacterized to date. Herein we show that it, like Nopp140, is a highly phosphorylated nucleolar protein. However, treacle fails to colocalize with Nopp140 to Cajal (coiled) bodies. As in the case of Nopp140, casein kinase 2 appears to be responsible for the unusually high degree of phosphorylation as evidenced by its coimmunoprecipitation with treacle. Based on these and other observations, treacle and Nopp140 exhibit distinct but overlapping functions. The majority of TCOF1 mutations in TCS lead to premature termination codons that could affect the cellular levels of the full-length treacle. We demonstrate however, that the cellular amount of treacle varies less than twofold among a collection of primary fibroblasts and lymphoblasts and regardless of whether the cells were derived from TCS patients or healthy individuals. Therefore, cells of TCS patients possess a mechanism to maintain wild-type levels of full-length treacle from a single allele.

Regional cortical white matter reductions in velocardiofacial syndrome: a volumetric MRI analysis.

BACKGROUND: Velocardiofacial syndrome, caused by a microdeletion on chromosome 22q.11, is associated with craniofacial anomalies, cardiac defects, learning disabilities, and psychiatric disorders. To understand how the 22q.11 deletion affects brain development, this study examined gray and white matter volumes in major lobar brain regions of children with velocardiofacial syndrome relative to control subjects. METHODS: Subjects were ten children with velocardiofacial syndrome and ten age- and gender-matched unaffected children. Coronal images were acquired with a 3-D spoiled gradient echo series and partitioned into 124, 1.5-mm contiguous slices. A stereotaxic grid was used to subdivide brain tissue into cerebral lobes, which were segmented into gray, white, and CSF compartments using an algorithm based on intensity values and tissue boundaries. Nonparametric statistics were used to compare lobar volumes of gray and white matter. RESULTS: Analyses indicated that children with velocardiofacial syndrome had significantly smaller volumes in nonfrontal, but not frontal, lobar brain regions. Volume reductions affected nonfrontal white matter to a greater extent than nonfrontal gray matter. CONCLUSIONS: The presence of white matter reductions may be related to disturbances in myelination or axonal integrity in velocardiofacial syndrome. Further work is required to delineate the nature and extent of white matter anomalies, and to link them to variation in the neurocognitive and neuropsychiatric phenotype of velocardiofacial syndrome.

Human cDNA clones transcribed from an unusually high-molecular-weight RNA encode a new collagen chain.

Human collagen (COL) cDNA clones were isolated from a library representing transcripts synthesized by an established rhabdomyosarcoma (RH) cell line. The 0.6-kb insert of the first isolate encodes a discontinuous collagenous sequence not homologous to type I-XVI COL chains. Sequencing of a second clone with a 4-kb insert revealed an open reading frame (ORF) of 2154 nucleotides. The deduced amino acid (aa) sequence begins with an 186-aa noncollagenous region containing seven cysteines (Cys). Several of the Cys and surrounding aa residues can be aligned with those in type XVI, XII and IX COL. Due to the presence of two long interruptions, the 524-aa collagenous region is separated into three subdomains that each have smaller interruptions of 1-6 aa. The protein terminates with an 8-aa noncollagenous peptide including an unusual single Cys which would be expected to form an interchain disulfide bond. Results of Northern blot hybridization suggest that the new COL chain may be uncommonly large since the clone identified a low-abundance RNA at least 12.4 kb in size. The gene coding for RH COL is located on human chromosome 6. It is now important to elucidate the role of this unusual COL in the infrastructure of extracellular matrix.

Cloning and chromosomal localization of the human BARX2 homeobox protein gene.

The human BARX2 gene encodes a homeodomain-containing protein of 254 amino acids, which binds optimally to the DNA consensus sequence YYTAATGRTTTTY. BARX2 is highly expressed in adult salivary gland and is expressed at lower levels in other tissues, including mammary gland, kidney, and placenta. The BARX2 gene consists of four exons, and is located on human chromosome 11q25. This chromosomal location is within the minimal deletion region for Jacobsen syndrome, a syndrome including craniosynostosis and other developmental abnormalities. This chromosomal location, along with the reported expression of murine barx2 in craniofacial development, suggests that BARX2 may be causally involved in the craniofacial abnormalities in Jacobsen syndrome.

A human synaptic vesicle monoamine transporter cDNA predicts posttranslational modifications, reveals chromosome 10 gene localization and identifies TaqI RFLPs.

A human vesicular monoamine transporter cDNA has been identified by screening a human brainstem library using sequences from the rat brain synaptic vesicle monoamine transporter (SVMT) [(1992) Cell 70, 539-551; (1992) Proc. Natl. Acad. Sci. USA 89, 10993-10997]. The hSVMT shares 92% amino acid identity with the rat sequence, but displays one less consensus site for asparagine N-linked glycosylation and one more consensus site for phosphorylation by protein kinase C. The human SVMT gene maps to chromosome 10q25 using Southern blotting analysis of human/rodent hybrid cell lines and fluorescent in situ hybridization approaches. The cDNA, and a subclone, recognize TaqI polymorphisms that may prove useful to assess this gene's involvement in neuropsychiatric disorders involving monoaminergic brain systems.

Development of a microsatellite genetic map spanning 5q31-q33 and subsequent placement of the LGMD1A locus between D5S178 and IL9.

Limb-girdle muscular dystrophy (LGMD) is a genetically and clinically heterogeneous group of disorders. We previously localized an autosomal dominant form of the disorder (LGMD1A) to chromosome 5q22-31 by linkage analysis in a single large pedigree. After developing a microsatellite genetic map incorporating six loci in q31-33 of chromosome 5 and spanning 35 cM, we have refined the original localization. Using multipoint analysis, LGMD1A is localised to a 7 cM region between the markers IL9 and D5S178 with odds > 1000:1.

Mild mandibulofacial dysostosis in a child with a deletion of 3p.

We report on a patient with mild mandibulofacial dysostosis and a small interstitial deletion of 3p, 46,XY,del(3)(p23p24.12). Linkage of Treacher Collins syndrome, the most common of the mandibulofacial dysostoses, to the 5q31.3-->q33.3 region of chromosome 5 has been established. This is the fourth report of a patient with mandibulofacial dysostosis with a chromosome abnormality outside the 5q31.3 area. Mandibulofacial dysostosis is a heterogeneous entity, and evaluation and counseling of affected individuals should be undertaken with caution.

Two craniosynostotic patients with 11q deletions, and review of 48 cases.

Many chromosomal abnormalities have craniofacial manifestations. One such abnormality, partial monosomy of chromosome 11q, is associated with metopic synostosis and resultant trigonocephaly. We reviewed 48 published cases of 11q deletions and translocations. Eighty percent were associated with abnormal head shape. Also commonly found were hypertelorism, ptosis of the eyelids, wide or low nasal bridge, apparently low-set malformed ears, down-turned mouth, micro/retrognathia, digital and cardiac anomalies, and psychomotor retardation. We report on two patients referred for abnormal head shape. The first case had brachycephaly, flat occiput, hypertelorism, and maxillary hypoplasia. Karyotype was 46,XY,del(11)(q24.1-->qter). The second patient had trigonocephaly, hypotelorism, posteriorly angulated ears, horizontal crease below his lower lip, syndactyly, shawl scrotum, cryptorchidism, and inguinal hernias. Karyotype showed partial trisomy of chromosome 4q as well as partial monosomy of 11q [46,XY,11,+der(11)t(4;11) (q31.3;q25)], a combination not previously reported. Deletions of 11q appear to produce a wide spectrum of abnormalities.

Partial gonadal dysgenesis in a patient with a marker Y chromosome.

We evaluated a patient with partial gonadal dysgenesis including a right dysgenetic testis and a left streak gonad with rudimentary fallopian tube and uterus. She had ambiguous external genitalia and was raised female. Although her height is normal (25th centile at age 12 years), she has some findings of Ullrich-Turner syndrome. Her karyotype was reported to be 46,X,+marker; subsequent molecular investigations showed the marker to be the short arm of the Y chromosome. Genomic DNA, isolated from leukocytes of the patient and her father, was digested with a variety of restriction endonucleases and subjected to Southern blot analysis. A positive hybridization signal was obtained with probes for the short arm of the Y chromosome (pRsY0.55, SRY, ZFY, 47Z, pY-190, and YC-2) in DNA from the patient, indicating the presence of most if not all of the short arm, while long arm probes (HinfA and pY3.4) indicated that at least 75% of the long arm of the Y chromosome was missing. The gene responsible for testicular determination (TDF) is on the distal portion of the short arm of the Y chromosome; Yq has no known influence on sex determination. Hence, the deletion of the long arm of the Y chromosome cannot explain the gonadal dysgenesis in this patient. One explanation for the gonadal dysgenesis and Ullrich-Turner phenotype in the patient could be undetected 45,X/46,X,+marY mosaicism but no such mosaicism was observed in peripheral lymphocytes. Several investigators have suggested the presence of an "anti-Turner" gene near TDF. Hence it is possible that the clinical phenotype in our patient results from a Y chromosomal defect in sequences flanking TDF, which reduces the function of both TDF and the "anti-Turner" genes.

Clinical, cytogenetic, and fluorescence in situ hybridization findings in two cases of "complete ring" syndrome.

The term "ring syndrome" was proposed to describe a phenotype of growth failure without major malformations due to a ring autosome. The growth failure is thought to be caused by instability of the ring chromosome leading to aneusomy and cell death. Most previous studies of ring chromosomes were based on standard cytogenetic banding techniques and were limited to microscopically detectable deletions in the ring chromosomes. We report on two patients with complete ring (4) and ring (9) chromosomes, respectively. The first was a 15-month-old girl and the second was a 16-month-old boy. They both presented with severe, symmetrical growth failure and normal psychomotor development in the absence of malformations. Their parents had a normal phenotype. The first case had a whorled pattern of hyperpigmentation and hypopigmentation on part of the face and chest, and the second case had a patchy hyperpigmented rash on the trunk. Peripheral blood karyotype of the first patient was 46,XX, r(4)(p16.3q35.2) and of the second 45,XY,-9/46,XY,r(9)(p24q34.3). G-band analysis suggested no loss of material in the ring chromosomes. These findings were confirmed by fluorescence in situ hybridization (FISH) analysis using chromosome-specific subtelomeric probes. The common human telomeric sequences were intact in the first patient but absent in the second patient. The cytogenetic and FISH data in our two cases provide further evidence for the existence of a "complete ring" phenotype independent of the autosome involved. Pigmentary skin changes are a useful clinical sign of mosaicism caused by the ring instability.

Syndrome of coronal craniosynostosis, Klippel-Feil anomaly, and sprengel shoulder with and without Pro250Arg mutation in the FGFR3 gene.

A unique Pro250Arg point mutation in fibroblast growth factor receptor 3 (FGFR3) was initially reported by Bellus et al. [1996: Nat Genet 14:174-176] and the phenotype subsequently by Muenke et al. [1997: Am J Hum Genet 60:555-564], Reardon et al. [1997: J Med Genet 34:632-636], and Graham et al. [1998: Am J Med Genet 77:322-329]. These authors emphasized the pleiotropic nature of this form of coronal craniosynostosis, including brachydactyly with carpal and/or tarsal coalitions, with other anomalies at lower frequency. We report on a family with autosomal dominant coronal synostosis, segmentation and fusion anomalies of the vertebra and ribs, and Sprengel shoulder due to the Pro250Arg mutation. We also report a single case with an identical phenotype without the mutation.

Saethre-Chotzen syndrome with familial translocation at chromosome 7p22.

Chromosome analysis of a male infant and his mother with Saethre-Chotzen syndrome demonstrated an apparently balanced translocation, t(2;7)(p23;p22). This association lends support to localization of the gene for Saethre-Chotzen syndrome to the 7p2 region and supports further involvement of gene(s) in the 7p22 region.