Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome.

Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator of the Rab3 pathway implicated in exocytic release of neurotransmitters and hormones, in 12 families with Micro syndrome. We hypothesize that the underlying pathogenesis of Micro syndrome is a failure of exocytic release of ocular and neurodevelopmental trophic factors.

Duplications involving a conserved regulatory element downstream of BMP2 are associated with brachydactyly type A2.

Autosomal-dominant brachydactyly type A2 (BDA2), a limb malformation characterized by hypoplastic middle phalanges of the second and fifth fingers, has been shown to be due to mutations in the Bone morphogenetic protein receptor 1B (BMPR1B) or in its ligand Growth and differentiation factor 5 (GDF5). A linkage analysis performed in a mutation-negative family identified a novel locus for BDA2 on chromosome 20p12.3 that incorporates the gene for Bone morphogenetic protein 2 (BMP2). No point mutation was identified in BMP2, so a high-density array CGH analysis covering the critical interval of approximately 1.3 Mb was performed. A microduplication of approximately 5.5 kb in a noncoding sequence approximately 110 kb downstream of BMP2 was detected. Screening of other patients by qPCR revealed a similar duplication in a second family. The duplicated region contains evolutionary highly conserved sequences suggestive of a long-range regulator. By using a transgenic mouse model we can show that this sequence is able to drive expression of a X-Gal reporter construct in the limbs. The almost complete overlap with endogenous Bmp2 expression indicates that a limb-specific enhancer of Bmp2 is located within the identified duplication. Our results reveal an additional functional mechanism for the pathogenesis of BDA2, which is duplication of a regulatory element that affects the expression of BMP2 in the developing limb.

Brachydactyly type A2 associated with a defect in proGDF5 processing.

We investigated a family with a brachydactyly type A2 and identified a heterozygous arginine to glutamine (R380Q) substitution in the growth/differentiation factor 5 (GDF5) in all affected individuals. The observed mutation is located at the processing site of the protein, at which the GDF5 precursor is thought to be cleaved releasing the mature molecule from the prodomain. In order to test the effect of the mutation, we generated the GDF5-R380Q mutant and a cleavage-resistant proGDF5 mutant (R380A/R381A) in vitro. Both mutants were secreted from chicken micromass cultures, but showed diminished biological activity. Western blot analyses showed that wt GDF5 was processed by the chicken micromass cells, whereas the mutants were not, indicating that the mutations interfere with processing and that this leads to a strong reduction of biological activity. To test the requirements for GDF5 processing in vitro we produced recombinant human (rh) proGDF5 wild-type protein in Escherichia coli. The results show that unprocessed (rh) proGDF5 is virtually inactive but can be proteolytically activated by different enzymes such as trypsin, furin, and MMP3. (rh) proGDF5 could thus be used as a locally administered depot form with retarded release of activity. In contrast to mature rhGDF5, (rh) proGDF5 shows a high solubility at physiological pH, a characteristic that might be useful for therapeutic applications.

Compound heterozygous ASPM mutations in Pakistani MCPH families.

Autosomal recessive primary microcephaly (MCPH) is characterized by reduced head circumference (50% of all reported families. In spite of the high frequency of MCPH in Pakistan only one case of compound heterozygosity for mutations in ASPM has been reported yet. In this large MCPH study we ascertained 37 families including 319 persons (140 patients). Haplotype analysis of eight STS markers suggested linkage by homozygosity in 20 families, and re-analysis of single sib ships in the remaining families demonstrated possible compound heterozygosity in two families. Direct sequencing indeed confirmed compound heterozygosity in two and homozygous mutations in 20 families, respectively, showing that up to 10% of families with MCPH caused by ASPM are compound heterozygous. In total we identified 16 different nonsense or frameshift mutations of which 12 were novel thereby increasing the number of mutations in ASPM significantly from 35 to 47. We found no correlation between the severity of the condition and the site of truncation. We suggest that the high frequency of compound heterozygosity observed in this study is taken into consideration as part of future genetic testing and counseling in Pakistani MCPH families.

Activating and deactivating mutations in the receptor interaction site of GDF5 cause symphalangism or brachydactyly type A2.

Here we describe 2 mutations in growth and differentiation factor 5 (GDF5) that alter receptor-binding affinities. They cause brachydactyly type A2 (L441P) and symphalangism (R438L), conditions previously associated with mutations in the GDF5 receptor bone morphogenetic protein receptor type 1b (BMPR1B) and the BMP antagonist NOGGIN, respectively. We expressed the mutant proteins in limb bud micromass culture and treated ATDC5 and C2C12 cells with recombinant GDF5. Our results indicated that the L441P mutant is almost inactive. The R438L mutant, in contrast, showed increased biological activity when compared with WT GDF5. Biosensor interaction analyses revealed loss of binding to BMPR1A and BMPR1B ectodomains for the L441P mutant, whereas the R438L mutant showed normal binding to BMPR1B but increased binding to BMPR1A, the receptor normally activated by BMP2. The binding to NOGGIN was normal for both mutants. Thus, the brachydactyly type A2 phenotype (L441P) is caused by inhibition of the ligand-receptor interaction, whereas the symphalangism phenotype (R438L) is caused by a loss of receptor-binding specificity, resulting in a gain of function by the acquisition of BMP2-like properties. The presented experiments have identified some of the main determinants of GDF5 receptor-binding specificity in vivo and open new prospects for generating antagonists and superagonists of GDF5.

Blue eye color in humans may be caused by a perfectly associated founder mutation in a regulatory element located within the HERC2 gene inhibiting OCA2 expression.

The human eye color is a quantitative trait displaying multifactorial inheritance. Several studies have shown that the OCA2 locus is the major contributor to the human eye color variation. By linkage analysis of a large Danish family, we finemapped the blue eye color locus to a 166 Kbp region within the HERC2 gene. By association analyses, we identified two SNPs within this region that were perfectly associated with the blue and brown eye colors: rs12913832 and rs1129038. Of these, rs12913832 is located 21.152 bp upstream from the OCA2 promoter in a highly conserved sequence in intron 86 of HERC2. The brown eye color allele of rs12913832 is highly conserved throughout a number of species. As shown by a Luciferase assays in cell cultures, the element significantly reduces the activity of the OCA2 promoter and electrophoretic mobility shift assays demonstrate that the two alleles bind different subsets of nuclear extracts. One single haplotype, represented by six polymorphic SNPs covering half of the 3' end of the HERC2 gene, was found in 155 blue-eyed individuals from Denmark, and in 5 and 2 blue-eyed individuals from Turkey and Jordan, respectively. Hence, our data suggest a common founder mutation in an OCA2 inhibiting regulatory element as the cause of blue eye color in humans. In addition, an LOD score of Z = 4.21 between hair color and D14S72 was obtained in the large family, indicating that RABGGTA is a candidate gene for hair color.

A novel nonsense mutation in MYO6 is associated with progressive nonsyndromic hearing loss in a Danish DFNA22 family.

Autosomal dominant inheritance is described in about 20% of all nonsyndromic hearing loss with currently 54 distinct loci (DFNA1-54), and >20 different genes identified. Seven different unconventional myosin genes are involved in ten different types of syndromic and nonsyndromic hearing loss with different patterns of inheritance: MYO7A in DFNA11/DFNB2/USH1B, MYH9 in DFNA17, MYH14 in DFNA4, MYO6 in DFNA22/DFNB37, MYO3A in DFNB30, MYO1A in DFNA48, and MYO15A in DFNB3. Two missense mutations in MYO6 (p.C442Y and p.H246R) have been characterized in families of Italian and American Caucasian extraction with autosomal dominant hearing loss, respectively, and the latter was associated with cardiomyopathy in some patients. Three Pakistani families had homozygosity for three MYO6 mutations (c.36insT, p.R1166X, and p.E216V, respectively), and was in one instance associated with retinal degeneration. In the present study, we linked autosomal dominant hearing loss in a large Danish family to a 38.9 Mb interval overlapping with the DFNA22/DFNB37 locus on chromosome 6q13. A novel nonsense mutation in MYO6 exon 25 (c.2545C > T; p.R849X) was identified in the family. The mutation co-segregated with the disease and the mutant allele is predicted to encode a truncated protein lacking the coiled-coil and globular tail domains. These domains are hypothesized to be essential for targeting myosin VI to its cellular compartments. No other system was involved indicating nonsyndromic loss. In conclusion, a novel nonsense MYO6 mutation causes post-lingual, slowly progressive autosomal dominant nonsyndromic moderate to severe hearing loss in a Danish family.

Mutation of the Planar Cell Polarity Gene VANGL1 in Adolescent Idiopathic Scoliosis.

STUDY DESIGN: Mutation analysis of a candidate disease gene in a cohort of patients with moderate to severe Adolescent idiopathic scoliosis (AIS). OBJECTIVE: To investigate if damaging mutations in the planar cell polarity gene VANGL1 could be identified in AIS patients. SUMMARY OF BACKGROUND DATA: AIS is a spinal deformity which occurs in 1% to 3% of the population. The cause of AIS is often unknown, but genetic factors are important in the etiology. Rare variants in genes encoding regulators of WNT/planar cell polarity (PCP) signaling were recently identified in AIS patients. METHODS: We analyzed the coding region of the VANGL1 gene for mutations using Sanger sequencing in 157 unrelated patients with moderate to severe AIS. The frequency of mutations in the patient cohort was compared with their frequency in a large cohort of controls. Functional effect of mutations were predicted in silico and analyzed in vitro by transfection of normal and mutant recombinant VANGL1 protein in Madin-Darby Canine Kidney (MDCK) cells. Cellular localization of recombinant proteins was analyzed by immunofluorescence microscopy analysis. RESULTS: In the patient cohort, we identified two rare missense mutations in VANGL1, encoding a receptor involved in WNT/PCP signaling. The mutations, p.I136N and p.F440 V, are very rare in the normal population. Both mutations are predicted to be damaging, and to affect evolutionary conserved amino acid residues of VANGL1. Functional analysis in MDCK cells showed that the mutations abolished the normal translocation of VANGL1 to the cell membrane. CONCLUSION: Our data support that mutations in genes involved in WNT/PCP signaling may be associated with AIS, but replication in other patient cohorts and further analysis of the role of WNT/PCP signaling in AIS is needed. LEVEL OF EVIDENCE: 4.

Germline LEMD3 mutations are rare in sporadic patients with isolated melorheostosis.

To further explore the allelic heterogeneity within the group of LEMD3-related disorders, we have screened a larger series of patients including 5 probands with osteopoikilosis or Buschke-Ollendorff syndrome (BOS), 2 families with the co-occurrence of melorheostosis and BOS, and 12 unrelated patients with isolated melorheostosis. Seven novel LEMD3 mutations were identified, all predicted to result in loss-of-function of the protein. We confirm that loss-of-function mutations in the LEMD3 gene can result in either osteopoikilosis or BOS. However, LEMD3 germline mutations were only found in two melorheostosis patients belonging to a different BOS family and one sporadic patient with melorheostosis. The additional presence of osteopoikilosis lesions in these patients seemed to distinguish them from the group of sporadic melorheostosis patients where no germline LEMD3 mutation was identified. Somatic mosaicism for a LEMD3 mutation in the latter group was also not observed, and therefore we must conclude that the genetic defect in the majority of sporadic and isolated melorheostosis remains unknown.

Delineation of the ADULT syndrome phenotype due to arginine 298 mutations of the p63 gene.

The ADULT syndrome (Acro-Dermato-Ungual-Lacrimal-Tooth, OMIM 103285) is a rare ectodermal dysplasia associated with limb malformations and caused by heterozygous mutations in p63. ADULT syndrome has clinical overlap with other p63 mutation syndromes, such as EEC (OMIM 604292), LMS (OMIM 603543), AEC (106260), RHS (129400) and SHFM4 (605289). ADULT syndrome characteristics are ectrodactyly, ectodermal dysplasia, mammary gland hypoplasia and normal lip and palate. The latter findings allow differentiation from EEC syndrome. LMS differs by milder ectodermal involvement. Here, we report three new unrelated ADULT syndrome families, all with mutations of arginine 298. On basis of 16 patients in five families with R298 mutation, we delineate the ADULT syndrome phenotype. In addition, we have documented a gain-of-function effect on the dNp63gamma isoform caused by this mutation. We discuss the possible relevance of oral squamous cell carcinoma in one patient, who carries this p63 germline mutation.

Limb anomalies: Developmental and evolutionary aspects.

In this review we describe the developmental mechanisms involved in the making of a limb, by focusing on the nature and types of interactions of the molecules that play a part in the regulation of limb patterning and characterizing clinical conditions that are known to result from the abnormal function of these molecules. The latter subject is divided into sections dealing with syndromal and nonsyndromal deficiencies, polydactylies, and brachydactylies. Conditions caused by mutations in homeobox genes and fibroblast growth factors and their receptor genes are listed separately. Since the process of limb development has been conserved for more than 300 millions years, with all the necessary adaptive modifications occurring throughout evolution, we also take into consideration the evolutionary aspects of limb development in terms of genetic repertoire, molecular pathways, and morphogenetic events.

Preaxial polydactyly/triphalangeal thumb is associated with changed transcription factor-binding affinity in a family with a novel point mutation in the long-range cis-regulatory element ZRS.

A cis-regulatory sequence also known as zone of polarizing activity (ZPA) regulatory sequence (ZRS) located in intron 5 of LMBR1 is essential for expression of sonic hedgehog (SHH) in the developing posterior limb bud mesenchyme. Even though many point mutations causing preaxial duplication defects have been reported in ZRS, the underlying regulatory mechanism is still unknown. In this study, we analyzed the effect on transcription factor binding of a novel ZRS point mutation (463T>G) in a Pakistani family with preaxial polydactyly and triphalangeal thumb. Electrophoretical mobility shift assay demonstrated a marked difference between wild-type and the mutant probe, which uniquely bound one or several transcription factors extracted from Caco-2 cells. This finding supports a model in which ectopic anterior SHH expression in the developing limb results from abnormal binding of one or more transcription factors to the mutant sequence.

Suggestive linkage to a neighboring region of IRF6 in a cleft lip and palate multiplex family.

Cleft lip and/or palate (CL/P) is a common congenital malformation with a complex etiology, as many genes and environmental factors have been shown to play a role in craniofacial development. We used a genetic mapping approach to analyze a family with multiplex CL/P. A genome-wide scan with a 10 kb single nucleotide polymorphism (SNP) chip followed by fine mapping with microsatellite markers in a CL/P multiplex family suggested linkage (maximum multipoint LOD score of 2.41) to a 6.5 Mb interval at 1q32.1-q32.2. This interval was close to, but excluded IRF6. Mutations in the IRF6 (1q32.2) cause syndromic forms of CL/P, and several association studies have shown that polymorphisms in and around IRF6 are associated with non-syndromic CL/P (NSCLP). However, in the family described here, IRF6 was excluded from the linkage interval. Sequencing of selected genes in the interval and comparative genome hybridization (CGH) did not reveal any mutations or genomic aberrations. Our data suggest that an unidentified CL/P gene, or a non-coding IRF6 regulatory element in this linkage interval may have caused CL/P in this family.

Immunohistochemical expression of p63 in human prenatal tooth primordia.

The aim of this study was to investigate the expression of the p63 gene in normal human tooth buds at different gestational stages. This is the first detailed study of p63 expression in normal human prenatal tooth primordia. The material consisted of sections of the midaxial tissue block from the cranial base of three human fetuses of gestational ages (GA) 11, 15, and 21 weeks. The sections included tooth primordia representing cap stages and bell stages of human tooth morphogenesis. In the present study, immunostaining was carried out using the primary antibody, monoclonal mouse anti human p63 protein, clone 4A4. The sections were counterstained with hematoxylin Mayer. p63 immunoreactivity was identified by microscopy. The study showed a positive reaction of p63 in both the cap stage and the bell stage. In both stages, positivity was observed in the cells of the oral mucosa, the inner and outer enamel epithelium, and in the primary and secondary dental lamina. In the early cap stage, there is a strong positive reaction to p63 in the enamel knot, but not in the late cap stage. We suggest that p63 may have an important regulatory function in the enamel knot.

Male-to-male transmission in Laurin-Sandrow syndrome and exclusion of RARB and RARG.

We report on a father and a son with nasal and limb defects characteristic of Laurin-Sandrow syndrome (LSS) excluding for the first time X-linked inheritance in this rare condition. Based on a search for genes expressed late during nose formation and early in limb formation we identified retinoic acid receptor B (RARB) and retinoic acid receptor G (RARG) as possible candidate genes and sequenced bidirectionally including all exons and intron-exon bounders. We identified a single nucleotide substitution in intron 2 of RARB, which is conserved in human, chimp, dog, mouse, rat, and chicken. However, it was located 83 bp from exon 2, suggesting it is a rare polymorphism which does not account for the phenotype. No other mutations were found. This suggests that another yet unknown gene is responsible for the condition.

A 72-year-old Danish puzzle resolved--comparative analysis of phenotypes in families with different-sized HOXD13 polyalanine expansions.

A phenotype-genotype correlation was previously described for carriers of different sized of polyalanine expansions in HOXD13. We report on a detailed comparison of 55 members (approximately 220 limbs) from 4 Danish families with duplications of 21 or 27 bp, expanding the polyalanine repeat from 15 to 22 and 24 residues, respectively. Two of these were previously described by Danish pioneers of human genetics, Tage Kemp and Oluf Thomsen. A clinical score was assigned to each limb based on manifestations assumed to represent different degrees of a duplication defect in hand rays 3-4 and foot rays 4-5. The length of metacarpals and phalangeal bones in rays 1, 2, and 5 was measured on hand radiographs and converted to Z-scores. The relative difference between corresponding right and left bones and directional, total, and fluctuating asymmetry was calculated for each individual. All of these parameters were compared between carriers of the +9 alanine expansion, the +7 alanine expansion, and non-mutation carriers with affected parents from the two families. Upper limb scores and the rate of abnormal bones (>2SD) were significantly higher in the first group than in the others. The first metacarpal and the middle phalanx of the little finger were significantly shorter, and the proximal phalanx of the index finger was significantly longer in this group than in the others. An increased level of total and fluctuating asymmetry was observed in long expansion carriers. Thus, our data have added evidence to the phenotype-genotype correlation previously reported, which was further extended to include lesser involvement of bones in ray 1, 2, and 5.

Sensorineural deafness, abnormal genitalia, synostosis of metacarpals and metatarsals 4 and 5, and mental retardation: description of a second patient and exclusion of HOXD13.

In 1988 Pfeiffer and Kapferer reported on a patient with sensorineural deafness, psychomotor delay, hypospadias, cerebral manifestations, and bilateral synostosis of the 4th and 5th metacarpals and metatarsals. Synostosis of the 4th and 5th metacarpals and metatarsals is a very rare defect that has been described as an isolated Mendelian defect, as part of multiple congenital anomaly (MCA) patterns, and in different syndromes. Among a total of 2,023,155 liveborn infants in the Spanish Collaborative Study of Congenital Malformations (ECEMC), we observed only two cases with this type of metacarpal fusion, for a frequency of 1/1,011,577. One had the isolated defect, and the other one that we are describing here, had an MCA pattern similar to that described by Pfeiffer and Kapferer [1988]. We tested HOXD13 but did not find any mutations in exons and intron-exon boundaries. To our knowledge this case is the second one reported with this syndrome.