Analysis of the p63 gene in classical EEC syndrome, related syndromes, and non-syndromic orofacial clefts.

EEC syndrome is an autosomal dominant disorder with the cardinal signs of ectrodactyly, ectodermal dysplasia, and orofacial clefts. EEC syndrome has been linked to chromosome 3q27 and heterozygous p63 mutations were detected in unrelated EEC families. In addition, homozygous p63 null mice exhibit craniofacial abnormalities, limb truncations, and absence of epidermal appendages, such as hair follicles and tooth primordia. In this study, we screened 39 syndromic patients, including four with EEC syndrome, five with syndromes closely related to EEC syndrome, and 30 with other syndromic orofacial clefts and/or limb anomalies. We identified heterozygous p63 mutations in three unrelated cases of EEC syndrome, two Iowa white families and one sporadic case in a Filipino boy. One family is atypical for EEC and has features consistent with Hay-Wells syndrome. In this family, the mutation ablates a splice acceptor site and, in the other two, mutations produce amino acid substitutions, R280C and R304Q, which alter conserved DNA binding sites. Germline mosaicism was detected in the founder of the mutation in one case. These three cases show significant interfamilial and intrafamilial variability in expressivity. We also screened p63 in 62 patients with non-syndromic orofacial clefts, identifying an intronic single nucleotide polymorphism but finding no evidence of mutations that would explain even a subset of non-syndromic orofacial clefts. This study supports a common role for p63 in classical EEC syndrome, both familial and sporadic, but not in other related or non-syndromic forms of orofacial clefts.

p63 Gene mutations in eec syndrome, limb-mammary syndrome, and isolated split hand-split foot malformation suggest a genotype-phenotype correlation.

p63 mutations have been associated with EEC syndrome (ectrodactyly, ectodermal dysplasia, and cleft lip/palate), as well as with nonsyndromic split hand-split foot malformation (SHFM). We performed p63 mutation analysis in a sample of 43 individuals and families affected with EEC syndrome, in 35 individuals affected with SHFM, and in three families with the EEC-like condition limb-mammary syndrome (LMS), which is characterized by ectrodactyly, cleft palate, and mammary-gland abnormalities. The results differed for these three conditions. p63 gene mutations were detected in almost all (40/43) individuals affected with EEC syndrome. Apart from a frameshift mutation in exon 13, all other EEC mutations were missense, predominantly involving codons 204, 227, 279, 280, and 304. In contrast, p63 mutations were detected in only a small proportion (4/35) of patients with isolated SHFM. p63 mutations in SHFM included three novel mutations: a missense mutation (K193E), a nonsense mutation (Q634X), and a mutation in the 3' splice site for exon 5. The fourth SHFM mutation (R280H) in this series was also found in a patient with classical EEC syndrome, suggesting partial overlap between the EEC and SHFM mutational spectra. The original family with LMS (van Bokhoven et al. 1999) had no detectable p63 mutation, although it clearly localizes to the p63 locus in 3q27. In two other small kindreds affected with LMS, frameshift mutations were detected in exons 13 and 14, respectively. The combined data show that p63 is the major gene for EEC syndrome, and that it makes a modest contribution to SHFM. There appears to be a genotype-phenotype correlation, in that there is a specific pattern of missense mutations in EEC syndrome that are not generally found in SHFM or LMS.

Identification and characterization of an Xq26-q27 duplication in a family with spina bifida and panhypopituitarism suggests the involvement of two distinct genes.

We investigated a family with a duplication, dup(X)q26-q27, that was present in two brothers, their mother, and their maternal grandmother. The brothers carrying the duplication displayed spina bifida and panhypopituitarism, whereas a third healthy brother inherited the normal X chromosome. Preferential inactivation of the X chromosome containing the duplication was evident in healthy carrier females. We determined the boundaries of the Xq26-q27 duplication. Via interphase FISH analysis we narrowed down each of the two breakpoint regions to approximately 300-kb intervals. The proximal breakpoint is located in Xq26.1 between DXS1114 and HPRT and is contained in YAC yWXD599, while the distal breakpoint is located in Xq27.3 between DXS369 and DXS1200 and contained in YAC yWXD758. The duplication comprises about 13 Mb. Evidence from the literature points to a predisposing gene for spina bifida in Xq27. We hypothesize that the spina bifida in the two brothers may be due to interruption of a critical gene in the Xq27 breakpoint region. Several candidate genes were mapped to the Xq27 critical region but none was shown to be disrupted by the duplication event. Recently, M. Lagerström-Fermér et al. (1997, Am. J. Hum. Genet. 60, 910-916) reported on a family with X-linked recessive panhypopituitarism associated with a duplication in Xq26; however, no details were reported on the extent of the duplication. Our study corroborates their hypothesis that X-linked recessive panhypopituitarism is likely to be caused by a gene encoding a dosage-sensitive protein involved in pituitary development. We place the putative gene between DXS1114 and DXS1200, corresponding to the interval defined by the duplication in the present family.

Study on the gene and phenotypic characterisation of autosomal recessive demyelinating motor and sensory neuropathy (Charcot-Marie-Tooth disease) with a gene locus on chromosome 5q23-q33.

OBJECTIVES: To report the occurrence of the autosomal recessive form of demyelinating Charcot-Marie-Tooth disease (CMT) with a locus on chromosome 5q23-33 in six non-related European families, to refine gene mapping, and to define the disease phenotype. METHODS: In an Algerian patient with autosomal recessive demyelinating CMT mapped to chromosome 5q23-q33 the same unique nerve pathology was established as previously described in families with a special form of autosomal recessive demyelinating CMT. Subsequently, the DNA of patients with this phenotype was tested from five Dutch families and one Turkish family for the 5q23-q33 locus. RESULTS: These patients and the Algerian families showed a similar and highly typical combination of clinical and morphological features, suggesting a common genetic defect. A complete cosegregation for markers D5S413, D5S434, D5S636, and D5S410 was found in the families. Haplotype construction located the gene to a 7 cM region between D5S643 and D5S670. In the present Dutch families linkage disequilibrium could be shown for various risk alleles and haplotypes indicating that most of these families may have inherited the underlying genetic defect form a common distant ancestor. CONCLUSIONS: This study refines the gene localisation of autosomal recessive demyelinating CMT, mapping to chromosome 5q23-33 and defines the phenotype characterised by a precocious and rapidly progressive scoliosis in combination with a relatively mild neuropathy and a unique pathology. Morphological alterations in Schwann cells of the myelinated and unmyelinated type suggest the involvement of a protein present in both Schwann cell types or an extracellular matrix protein rather than a myelin protein. The combination of pathological features possibly discerns autosomal recessive demyelinating CMT with a gene locus on chromosome 5q23-33 from other demyelinating forms of CMT disease.

Refined genetic and physical localization of the Wagner disease (WGN1) locus and the genes CRTL1 and CSPG2 to a 2- to 2.5-cM region of chromosome 5q14.3.

Wagner syndrome (WGN1; MIM 143200), an autosomal dominant vitreoretinopathy characterized by chorioretinal atrophy, cataract, and retinal detachment, is linked to 5q14.3. Other vitreoretinopathies without systemic stigmata, including erosive vitreoretinopathy, are also linked to this region and are likely to be allelic. Within the critical region lie genes encoding two extracellular macromolecules, link protein (CRTL1) and versican (CSPG2), which are important in binding hyaluronan, a significant component of the mammalian vitreous gel, and which therefore represent excellent candidates for Wagner syndrome. Genetic mapping presented here in two further families reduces the critical region to approximately 2 cM. Subsequent refinement of the physical map allows ordering of known polymorphic microsatellites and excludes CRTL1 as a likely candidate for the disorder. CSPG2 is shown to lie within the critical region; however, analysis of the complete coding region of the mature peptide reveals no clear evidence that it is the gene underlying WGN1.

Limb mammary syndrome: a new genetic disorder with mammary hypoplasia, ectrodactyly, and other Hand/Foot anomalies maps to human chromosome 3q27.

We report on a large Dutch family with a syndrome characterized by severe hand and/or foot anomalies, and hypoplasia/aplasia of the mammary gland and nipple. Less frequent findings include lacrimal-duct atresia, nail dysplasia, hypohydrosis, hypodontia, and cleft palate with or without bifid uvula. This combination of symptoms has not been reported previously, although there is overlap with the ulnar mammary syndrome (UMS) and with ectrodactyly, ectodermal dysplasia, and clefting syndrome. Allelism with UMS and other related syndromes was excluded by linkage studies with markers from the relevant chromosomal regions. A genomewide screening with polymorphic markers allowed the localization of the genetic defect to the subtelomeric region of chromosome 3q. Haplotype analysis reduced the critical region to a 3-cM interval of chromosome 3q27. This chromosomal segment has not been implicated previously in disorders with defective development of limbs and/or mammary tissue. Therefore, we propose to call this apparently new disorder "limb mammary syndrome" (LMS). The SOX2 gene at 3q27 might be considered an excellent candidate gene for LMS because the corresponding protein stimulates expression of FGF4, an important signaling molecule during limb outgrowth and development. However, no mutations were found in the SOX2 open reading frame, thus excluding its involvement in LMS.

Altered regulation of platelet-derived growth factor receptor-alpha gene-transcription in vitro by spina bifida-associated mutant Pax1 proteins.

Mouse models show that congenital neural tube defects (NTDs) can occur as a result of mutations in the platelet-derived growth factor receptor-alpha gene (PDGFRalpha). Mice heterozygous for the PDGFRalpha-mutation Patch, and at the same time homozygous for the undulated mutation in the Pax1 gene, exhibit a high incidence of lumbar spina bifida occulta, suggesting a functional relation between PDGFRalpha and Pax1. Using the human PDGFRalpha promoter linked to a luciferase reporter, we show in the present paper that Pax1 acts as a transcriptional activator of the PDGFRalpha gene in differentiated Tera-2 human embryonal carcinoma cells. Two mutant Pax1 proteins carrying either the undulated-mutation or the Gln --> His mutation previously identified by us in the PAX1 gene of a patient with spina bifida, were not or less effective, respectively. Surprisingly, Pax1 mutant proteins appear to have opposing transcriptional activities in undifferentiated Tera-2 cells as well as in the U-2 OS osteosarcoma cell line. In these cells, the mutant Pax1 proteins enhance PDGFRalpha-promoter activity whereas the wild-type protein does not. The apparent up-regulation of PDGFRalpha expression in these cells clearly demonstrates a gain-of-function phenomenon associated with mutations in Pax genes. The altered transcriptional activation properties correlate with altered protein-DNA interaction in band-shift assays. Our data provide additional evidence that mutations in Pax1 can act as a risk factor for NTDs and suggest that the PDGFRalpha gene is a direct target of Pax1. In addition, the results support the hypothesis that deregulated PDGFRalpha expression may be causally related to NTDs.

Hereditary neuropathy with liability to pressure palsies. Phenotypic differences between patients with the common deletion and a PMP22 frame shift mutation.

In six families with hereditary neuropathy with liability to pressure palsies (HNPP) the 17p11.2 deletion was absent, but single strand conformation-analysis and subsequent sequencing demonstrated a heterozygous G-insertion in a stretch of six Gs at nt 276281 of the PMP22 gene, resulting in a frame shift after Gly94. Haplotype comparison of the six families revealed common ancestry. We compared the phenotype of 23 patients from these six families with the phenotype of 63 patients of 17 families with the common deletion. The patients with the G-insertion showed the clinical, electrophysiological and morphological characteristics of common HNPP, but in addition they had significantly more neuropathic features, mimicking hereditary motor and sensory neuropathy type I (HMSN I) or Charcot-Marie-Tooth disease type 1 (CMT1). To explain this distinct phenotype we suggest that, by translation of the mutated gene, a markedly changed polypeptide is formed without the normal cytoplasmic C-terminal of the native protein, resulting in a loss of function similar to that with the common deletion, but exerting an extra disturbance of Schwann cell functions, probably by hampering normal myelin formation or maintenance.

Two divergent types of nerve pathology in patients with different P0 mutations in Charcot-Marie-Tooth disease.

In seven unrelated patients with a demyelinating motor and sensory neuropathy, we found mutations in exons 2 and 3 of the P0 gene. Morphologic examination of sural nerve biopsy specimens showed a demyelinating process with onion bulb formation in all cases. In four patients, ultrastructural examination demonstrated uncompacted myelin in 23 to 68% of the myelinated fibers, which is in agreement with the widely accepted function of P0 as a homophilic adhesion molecule. Three patients showed normal compact myelin, but morphology was dominated by the abundant occurrence of focally folded myelin. The two divergent pathologic phenotypes exemplify that some mutations act differently on P0 protein formation or function than others, which is probably determined by site and nature of the mutation in the P0 gene.

Autosomal dominant and recessive osteochondrodysplasias associated with the COL11A2 locus.

Identifying mutations that cause specific osteochondrodysplasias will provide novel insights into the function of genes that are essential for skeletal morphogenesis. We report here that an autosomal dominant form of Stickler syndrome, characterized by mild spondyloepiphyseal dysplasia, osteoarthritis, and sensorineural hearing loss, but no eye involvement, is caused by a splice donor site mutation resulting in "in-frame" exon skipping within the COL11A2 gene, encoding the alpha 2(XI) chain of the quantitatively minor fibrillar collagen XI. We also show that an autosomal recessive disorder with similar, but more severe, characteristics is linked to the COL11A2 locus and is caused by a glycine to arginine substitution in alpha 2(XI) collagen. The results suggest that mutations in collagen XI genes are associated with a spectrum of abnormalities in human skeletal development and support the conclusion of others, based on studies of murine chondrodysplasia, that collagen XI is essential for skeletal morphogenesis.

Fine mapping of a putatively imprinted gene for familial non-chromaffin paragangliomas to chromosome 11q13.1: evidence for genetic heterogeneity.

Autosomal, dominantly inherited, non-chromaffin paragangliomas are tumors of the head and neck region occurring with a frequency of 1:30,000. Genomic imprinting probably influences the expression of the disorder, because tumor development is limited to individuals who have inherited the trait from their father. By linkage analysis and haplotyping of a single large family in which the pattern of inheritance is consistent with genomic imprinting, we have mapped the gene to a 5 cM region of chromosome 11q13.1 between D11S956 and PYGM. A maximum lod score of 7.62 at theta = 0.0 was obtained for D11S480. This interval does not overlap with a recently assigned locus for glomus tumors in other families: 11q22.3-q23.3. Furthermore, analysis of a second family showing the imprinting phenomenon resulted in the exclusion of the 5 cM area as the location of the disease gene, whereas an indication for linkage was obtained (Z = +2.65) with markers from the distal locus. These observations argue for the presence of two distinct imprinted genes for glomus tumors on 11q. A model for tumor initiation and progression is presented based on all available information.

Clinical and molecular studies in a large Dutch family with Noonan syndrome.

We describe the largest Noonan syndrome (NS) family reported to date. The manifestations of the affected relatives are discussed. In the absence of a biochemical marker NS is still a clinical diagnosis. The diagnostic criteria that were used are presented compared with other published criteria for diagnosing NS. The large size of this family enabled us to test the possible involvement of candidate regions by multipoint linkage analysis. Both the region surrounding the NF1 locus on chromosome 17 and the proximal part of chromosome 22 could be excluded. Since NS may well be heterogeneous, the use of such a large family in linkage studies of NS should prove indispensable.

Prevalence of the 1.5-Mb 17p deletion in families with hereditary neuropathy with liability to pressure palsies.

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder of the peripheral nerves leading to increased susceptibility to mechanical traction or compression. Some patients have been shown to be carriers of a 1.5-Mb deletion in chromosome 17p11.2, which corresponds to the duplicated region present in most patients with Charcot-Marie-Tooth disease type 1A. Recently, evidence has been presented that the deletion is not the only cause of HNPP. To determine the prevalence of the 1.5-Mb deletion, we have examined 22 unrelated families with HNPP in the following two ways: by polymerase chain reaction analysis of marker loci D17S122 and D17S261, and by gene dosage measurements with DNA probes for D17S122 (VAW409R3a) and D17S125 (VAW412R3a) and for the PMP-22 gene. The efficiency and sensitivity of these methods is discussed. Our results show that the prevalence of the 17p deletion in our families with HNPP is 68%. One patient, presenting as a sporadic case, was found to be affected by a de novo deletion in the paternal chromosome. Single-strand conformation analysis of the protein-coding region of the PMP-22 gene did not reveal any mutation in patients from the 7 families lacking the 17p deletion. As a group, these families could not be distinguished by clinical, electrophysiological, or morphological features from the families with the deletion.

A Stickler syndrome gene is linked to chromosome 6 near the COL11A2 gene.

Stickler syndrome (hereditary arthro-ophthalmopathy) is caused by mutations in the structural gene for collagen type II (COL2A1) in approximately 50% of cases. In the other families with this syndrome, the genetic defect is unknown. We have performed linkage analysis in a large Dutch kindred with a Stickler syndrome phenotype that was unlinked to COL2A1. As an initial strategy, we tested polymorphisms that are within or near genes encoding other cartilage collagens. Close linkage was demonstrated with polymorphic markers from 6p22 to 6p21.3. The highest lod score was 4.36 without recombination with D6S276. Since COL11A2 has also been localized to this chromosome region, a mutation in this collagen gene is an attractive explanation for the Stickler syndrome phenotype in this family. These data support the hypothesis that abnormalities of type XI collagen may be involved in inherited osteochondrodysplasias, such as Stickler syndrome.

Mental retardation, congenital heart defect, cleft palate, short stature, and facial anomalies: a new X-linked multiple congenital anomalies/mental retardation syndrome: clinical description and molecular studies.

We report on two brothers and their two maternal uncles with severe mental retardation, congenital heart defect, cleft or highly arched palate, short stature and craniofacial anomalies consisting of microcephaly, abnormal ears, bulbous nose, broad nasal bridge, malar hypoplasia, and micrognathia. Three of the four patients died at an early age. The mother of the two brothers had an atrial septal defect. She is assumed to be a manifesting carrier of a mutant gene, which is expressed in her two sons and two brothers. By multipoint linkage analysis it is found that the most likely location of the responsible gene is the pericentromeric region Xp21.3-q21.3 with DMD and DXS3 as flanking markers. Maximum information is obtained with marker DXS453 (Z = 1.20 at theta = 0.0).

Evidence for genetic heterogeneity underlying hereditary neuropathy with liability to pressure palsies.

Hereditary neuropathy with liability to pressure palsies (HNPP) is a disorder of the peripheral nervous system, the cause of which has recently been identified as a deletion on chromosome 17p. The deletion corresponds to the duplication that is commonly observed in patients with hereditary motor and sensory neuropathy type Ia (HMSNIa, 17p11.2-p12). Therefore, the gene for peripheral myelin protein 22 (PMP-22) is a candidate gene for both HMSNIa and HNPP. Here, we show that a similar deletion is present in one family with HNPP but is clearly absent in another family. Affected members of this family carry the expected two copies of the PMP-22 gene and the surrounding region. Furthermore, linkage analyses of this family exclude a large part of 17p, spanning the area deleted in other families with HNPP, as the location for the disease gene. These data strongly argue for the existence of genetic heterogeneity underlying HNPP. Results from two-point linkage analysis with markers on chromosome 1q are inconsistent with a possible involvement of the locus for HMSNIb in the present family.