An N-terminal G11A mutation in HOXD13 causes synpolydactyly and interferes with Gli3R function during limb pre-patterning.

Synpolydactyly (SPD) is a distal limb anomaly characterized by incomplete digit separation and the presence of supernumerary digits in the syndactylous web. This phenotype has been associated with mutations in the homeodomain or polyalanine tract of the HOXD13 gene. We identified a novel mutation (G11A) in HOXD13 that is located outside the previously known domains and affects the intracellular half life of the protein. Misexpression of HOXD13(G11A) in the developing chick limb phenocopied the human SPD phenotype. Finally, we demonstrated through in vitro studies that this mutation has a destabilizing effect on GLI3R uncovering an unappreciated mechanism by which HOXD13 determines the patterning of the limb.

Striking intrafamilial phenotypic variability in Aicardi-Goutières syndrome associated with the recurrent Asian founder mutation in RNASEH2C.

Aicardi-Goutières syndrome (AGS) is an encephalopathy of early childhood which is most commonly inherited as an autosomal recessive trait. The disorder demonstrates significant genetic heterogeneity with causative mutations in five genes identified to date. Although most patients with AGS experience a severe neonatal or infantile presentation, poor neurodevelopmental outcome and reduced survival, clinical variability in the onset and severity of the condition is being increasingly recognized. A later presentation with a more variable effect on development, morbidity and mortality has been particularly observed in association with mutations in SAMHD1 and RNASEH2B. In contrast, the recurrent c.205C > T (p.R69W) RNASEH2C Asian founder mutation has previously only been identified in children with a severe AGS phenotype. Here, to our knowledge, we present the first report of marked phenotypic variability in siblings both harboring this founder mutation in the homozygous state. In this family, one female child had a severe AGS phenotype with an onset in infancy and profound developmental delay, whilst an older sister was of completely normal intellect with a normal head circumference and was only diagnosed because of the presence of chilblains and a mild hemiplegia. An appreciation of intrafamilial phenotypic expression is important in the counseling of families considering prenatal diagnosis, and may also be relevant to the assessment of efficacy in future clinical trials. In addition, marked phenotypic variation raises the possibility that more mildly affected patients are not currently identified.

Carpenter syndrome: extended RAB23 mutation spectrum and analysis of nonsense-mediated mRNA decay.

Carpenter syndrome, a rare autosomal recessive disorder characterized by a combination of craniosynostosis, polysyndactyly, obesity, and other congenital malformations, is caused by mutations in RAB23, encoding a member of the Rab-family of small GTPases. In 15 out of 16 families previously reported, the disease was caused by homozygosity for truncating mutations, and currently only a single missense mutation has been identified in a compound heterozygote. Here, we describe a further 8 independent families comprising 10 affected individuals with Carpenter syndrome, who were positive for mutations in RAB23. We report the first homozygous missense mutation and in-frame deletion, highlighting key residues for RAB23 function, as well as the first splice-site mutation. Multi-suture craniosynostosis and polysyndactyly have been present in all patients described to date, and abnormal external genitalia have been universal in boys. High birth weight was not evident in the current group of patients, but further evidence for laterality defects is reported. No genotype-phenotype correlations are apparent. We provide experimental evidence that transcripts encoding truncating mutations are subject to nonsense-mediated decay, and that this plays an important role in the pathogenesis of many RAB23 mutations. These observations refine the phenotypic spectrum of Carpenter syndrome and offer new insights into molecular pathogenesis.

OBSL1 mutations in 3-M syndrome are associated with a modulation of IGFBP2 and IGFBP5 expression levels.

3-M syndrome is an autosomal recessive disorder characterized by severe pre- and postnatal growth retardation and minor skeletal changes. We have previously identified CUL7 as a disease-causing gene but we have also provided evidence of genetic heterogeneity in the 3-M syndrome. By homozygosity mapping in two inbred families, we found a second disease locus on chromosome 2q35-36.1 in a 5.2-Mb interval that encompasses 60 genes. To select candidate genes, we performed microarray analysis of cultured skin fibroblast RNA from one patient, looking for genes with altered expression; we found decreased expression of IGFBP2 and increased expression of IGFBP5. However, direct sequencing of these two genes failed to detect any anomaly. We then considered other candidate genes by their function/location and found nine distinct mutations in the OBSL1 gene in 13 families including eight nonsense and one missense mutations. To further understand the links between OBSL1, CUL7, and insulin-like growth factor binding proteins (IGFBPs), we performed real-time quantitative PCR (RT-PCR) analysis for OBSL1, CUL7, IGFBP2, and IGFBP5, using cultured fibroblast RNAs from two patients with distinct OBSL1 mutations (p.F697G; p.H814RfsX15). We found normal CUL7 mRNA levels but abnormal IGFBP2 and IGFBP5 mRNA levels in the two patients, suggesting that OBSL1 modulates the expression of IGFBP proteins.

Clinical and molecular genetic features of Beckwith-Wiedemann syndrome associated with assisted reproductive technologies.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a model imprinting disorder resulting from mutations or epigenetic events affecting imprinted genes at 11p15.5. Most BWS cases are sporadic and result from imprinting errors (epimutations) involving either of the two 11p15.5 imprinting control regions (IC1 and IC2). Previously, we and other reported an association between sporadic BWS and assisted reproductive technologies (ARTs). METHODS: In this study, we compared the clinical phenotype and molecular features of ART (IVF and ICSI) and non-ART children with sporadic BWS. A total of 25 patients with post-ART BWS were ascertained (12 after IVF and 13 after ICSI). RESULTS: Molecular genetic analysis revealed an IC2 epimutations (KvDMR1 loss of methylation) in 24 of the 25 children tested. Comparison of clinical features of children with post-ART BWS to those with non-ART BWS and IC2 defects revealed a lower frequency of exomphalos (43 versus 69%, P = 0.029) and a higher risk of neoplasia (two cases, P = 0.0014). As loss of methylation at imprinting control regions other than 11p15.5 might modify the phenotype of BWS patients with IC2 epimutations, we investigated differentially methylated regions (DMRs) at 6q24, 7q32 and 15q13 in post-ART and non-ART BWS IC2 cases (n = 55). Loss of maternal allele methylation at these DMRs occurred in 37.5% of ART and 6.4% of non-ART BWS IC2 defect cases. Thus, more generalized DMR hypomethylation is more frequent, but not exclusive to post-ART BWS. CONCLUSIONS: These findings provide further evidence that ART may be associated with disturbed normal genomic imprinting in a subset of children.

Clinical and radiological findings in Schinzel-Giedion syndrome.

The absence of a definitive genetic test for the autosomal recessive condition Schinzel-Giedion syndrome is a significant handicap to the recognition of this disorder. Radiological features have been an important aspect of many of the published cases. In a series of six cases, we now establish a consistency among many of the radiological features in affected cases which will be an important diagnostic aid in identifying future cases. This is confirmed by reference to an extensive review of previously published instances of the syndrome. Moreover, the clinical data, including previously unpublished photographs, which we detail from our patients will assist in enhanced diagnosis in the future.

Lacrimal outflow dysgenesis.

PURPOSE: To evaluate patients with lacrimal dysgenesis. DESIGN: Retrospective, nonrandomized, comparative case series. PARTICIPANTS: Fifty patients with lacrimal dysgenesis managed between 1992 and 2003. TESTING/INTERVENTION: The diagnosis of lacrimal outflow dysgenesis was made based on the following criteria: absent or hypoplastic punctum, canaliculus, lacrimal sac, and nasolacrimal duct, appearing in isolation or combination. Our management algorithm included observation, punctoplasty with intubation, or open lacrimal surgery, with or without intubation. MAIN OUTCOME MEASURE: Success, partial success, or failure of treatment. RESULTS: Lacrimal dysgenesis in our 50 patients (23 male and 27 female) involved 83 eyes, distributed as follows: proximal in 74 eyes (89%), distal in 27 (33%), and both in 18 (22%). Thirty (60%) patients presented with isolated lacrimal dysgenesis, and 20 (40%) presented with a systemic syndrome or dysmorphism. Thirty-three (66%) patients had bilateral involvement. Epiphora was the most common presenting symptom. Eighteen (36%) patients had a positive family history. Open lacrimal surgery was performed in a total of 29 (35%) of the 83 eyes: 25 had success, 3 had partial success, and 1 had failure. CONCLUSION: Both sporadic and hereditary forms of lacrimal outflow dysgenesis may present as an isolated finding or a part of a systemic syndrome or dysmorphism, occurring usually with bilateral involvement and presenting at a younger age in the setting of systemic anomalies. Proximal and distal lacrimal outflow systems may be involved with epiphora as the most common presenting symptom. The algorithm we have presented provides a systematic approach to the management of lacrimal outflow dysgenesis.

How genetically heterogeneous is Kabuki syndrome?: MLL2 testing in 116 patients, review and analyses of mutation and phenotypic spectrum.

MLL2 mutations are detected in 55 to 80% of patients with Kabuki syndrome (KS). In 20 to 45% patients with KS, the genetic basis remains unknown, suggesting possible genetic heterogeneity. Here, we present the largest yet reported cohort of 116 patients with KS. We identified MLL2 variants in 74 patients, of which 47 are novel and a majority are truncating. We show that pathogenic missense mutations were commonly located in exon 48. We undertook a systematic facial KS morphology study of patients with KS at our regional dysmorphology meeting. Our data suggest that nearly all patients with typical KS facial features have pathogenic MLL2 mutations, although KS can be phenotypically variable. Furthermore, we show that MLL2 mutation-positive KS patients are more likely to have feeding problems, kidney anomalies, early breast bud development, joint dislocations and palatal malformations in comparison with MLL2 mutation-negative patients. Our work expands the mutation spectrum of MLL2 that may help in better understanding of this molecule, which is important in gene expression, epigenetic control of active chromatin states, embryonic development and cancer. Our analyses of the phenotype indicates that MLL2 mutation-positive and -negative patients differ systematically, and genetic heterogeneity of KS is not as extensive as previously suggested. Moreover, phenotypic variability of KS suggests that MLL2 testing should be considered even in atypical patients.

Fourteen new cases contribute to the characterization of the 7q11.23 microduplication syndrome.

Interstitial deletions of 7q11.23 cause Williams-Beuren syndrome, one of the best characterized microdeletion syndromes. The clinical phenotype associated with the reciprocal duplication however is not well defined, though speech delay is often mentioned. We present 14 new 7q11.23 patients with the reciprocal duplication of the Williams-Beuren syndrome critical region, nine familial and five de novo. These were identified by either array-based MLPA or by array-CGH/oligonucleotide analysis in a series of patients with idiopathic mental retardation with an estimated population frequency of 1:13,000-1:20,000. Variable speech delay is a constant finding in our patient group, confirming previous reports. Cognitive abilities range from normal to moderate mental retardation. The association with autism is present in five patients and in one father who also carries the duplication. There is an increased incidence of hypotonia and congenital anomalies: heart defects (PDA), diaphragmatic hernia, cryptorchidism and non-specific brain abnormalities on MRI. Specific dysmorphic features were noted in our patients, including a short philtrum, thin lips and straight eyebrows. Our patient collection demonstrates that the 7q11.23 microduplication not only causes language delay, but is also associated with congenital anomalies and a recognizable face.

Mutations in the embryonal subunit of the acetylcholine receptor (CHRNG) cause lethal and Escobar variants of multiple pterygium syndrome.

Multiple pterygium syndromes (MPSs) comprise a group of multiple-congenital-anomaly disorders characterized by webbing (pterygia) of the neck, elbows, and/or knees and joint contractures (arthrogryposis). In addition, a variety of developmental defects (e.g., vertebral anomalies) may occur. MPSs are phenotypically and genetically heterogeneous but are traditionally divided into prenatally lethal and nonlethal (Escobar) types. To elucidate the pathogenesis of MPS, we undertook a genomewide linkage scan of a large consanguineous family and mapped a locus to 2q36-37. We then identified germline-inactivating mutations in the embryonal acetylcholine receptor gamma subunit (CHRNG) in families with both lethal and nonlethal MPSs. These findings extend the role of acetylcholine receptor dysfunction in human disease and provide new insights into the pathogenesis and management of fetal akinesia syndromes.

Management of myogenic ptosis.

OBJECTIVE: To review the genetics, clinical features, and management of patients affected by myogenic ptosis. DESIGN: Retrospective, noncomparative interventional case series. PARTICIPANTS: Twenty-eight patients with myogenic ptosis. METHODS: A review of all patients with myogenic ptosis between 1992 and 2000 was made in a tertiary oculoplastics practice. MAIN OUTCOME MEASURES: Ocular and systemic findings associated with myogenic ptosis were examined. Patients were diagnosed clinically, and ancillary tests (including genetic tests and muscle biopsy histologic findings) were reviewed. Surgical management principles and complications are discussed. RESULTS: Most of our ptosis patients had chronic progressive external ophthalmoplegia (43%), oculopharyngeal muscular dystrophy (OPMD; 18%), and myotonic dystrophy (18%). Fifty percent of myogenic ptosis patients in our series underwent frontalis suspensions. Twenty-one percent of patients who initially had operations at our institution had minor complications, most related to corneal exposure. The most common ocular finding other than ptosis and ophthalmoplegia was pigmentary retinopathy (25%). The most common systemic finding in our patients was dysphagia (43%). Genetic testing for OPMD was highly sensitive. Muscle biopsy results for mitochondrial myopathies were less accurate as adjunctive diagnostic tests. CONCLUSIONS: Myogenic ptosis should be considered in the differential diagnosis of any atypical ptosis presentation. To avoid complications, surgery should be performed only when the visual axis is obscured. We recommend the use of silicone slings in any patient with severe ptosis and less than 8 mm of levator function. Genetic testing of patients with myogenic ptosis is important to allow accurate diagnosis and to permit appropriate counseling on potentially life-threatening health issues.

Blepharophimosis: a recommendation for early surgery in patients with severe ptosis.

PURPOSE: To determine the optimal age for surgical correction of blepharophimosis. Associated features and their effects on incidence of amblyopia were also investigated. METHODS: The study was a retrospective case series of 28 patients with blepharophimosis, ptosis and epicanthus inversus syndrome presenting to a tertiary referral eyelid, lacrimal and orbital clinic. RESULTS: Amblyopia was present in 39% of patients. Patients with coexistent strabismus had a 64% incidence of amblyopia compared to 24% for those without strabismus. Hypermetropia was present in 43% of patients and 7% were myopic. Significant astigmatism was found in 40% of patients, but these factors did not increase the risk of amblyopia. Patients with severe ptosis had lower rates of amblyopia than those with moderate ptosis but had their ptosis corrected at a median age of 2 years compared to 5 years for those with moderate ptosis. There was an 18% incidence of nasolacrimal drainage problems. A good to excellent cosmetic outcome was achieved in 86% of patients. A positive family history was noted in 75% of patients, usually with paternal inheritance. CONCLUSIONS: Patients with blepharophimosis have a high rate of amblyopia. Co-existent strabismus doubles the risk of amblyopia. Ptosis alone causes mild to moderate amblyopia only. Patients with severe ptosis should have their ptosis corrected before 3 years of age, and all other patients should undergo surgery before 5 years of age.

FOXL2 and BPES: mutational hotspots, phenotypic variability, and revision of the genotype-phenotype correlation.

Blepharophimosis syndrome (BPES), an autosomal dominant syndrome in which an eyelid malformation is associated (type I) or not (type II) with premature ovarian failure (POF), has recently been ascribed to mutations in FOXL2, a putative forkhead transcription factor gene. We previously reported 22 FOXL2 mutations and suggested a preliminary genotype-phenotype correlation. Here, we describe 21 new FOXL2 mutations (16 novel ones) through sequencing of open reading frame, 5' untranslated region, putative core promoter, and fluorescence in situ hybridization analysis. Our study shows the existence of two mutational hotspots: 30% of FOXL2 mutations lead to polyalanine (poly-Ala) expansions, and 13% are a novel out-of-frame duplication. In addition, this is the first study to demonstrate intra- and interfamilial phenotypic variability (both BPES types caused by the same mutation). Furthermore, the present study allows a revision of the current genotype-phenotype correlation, since we found exceptions to it. We assume that for predicted proteins with a truncation before the poly-Ala tract, the risk for development of POF is high. For mutations leading to a truncated or extended protein containing an intact forkhead and poly-Ala tract, no predictions are possible, since some of these mutations lead to both types of BPES, even within the same family. Poly-Ala expansions may lead to BPES type II. For missense mutations, no correlations can be made yet. Microdeletions are associated with mental retardation. We conclude that molecular testing may be carefully used as a predictor for POF risk in a limited number of mutations.