Mutations in the LHX2 gene are not a frequent cause of micro/anophthalmia.

Purpose: Microphthalmia and anophthalmia are at the severe end of the spectrum of abnormalities in ocular development. A few genes (orthodenticle homeobox 2 [OTX2], retina and anterior neural fold homeobox [RAX], SRY-box 2 [SOX2], CEH10 homeodomain-containing homolog [CHX10], and growth differentiation factor 6 [GDF6]) have been implicated mainly in isolated micro/anophthalmia but causative mutations of these genes explain less than a quarter of these developmental defects. The essential role of the LIM homeobox 2 (LHX2) transcription factor in early eye development has recently been documented. We postulated that mutations in this gene could lead to micro/anophthalmia, and thus performed molecular screening of its sequence in patients having micro/anophthalmia. Methods: Seventy patients having non-syndromic forms of colobomatous microphthalmia (n=25), isolated microphthalmia (n=18), or anophthalmia (n=17), and syndromic forms of micro/anophthalmia (n=10) were included in this study after negative molecular screening for OTX2, RAX, SOX2, and CHX10 mutations. Mutation screening of LHX2 was performed by direct sequencing of the coding sequences and intron/exon boundaries. Results: Two heterozygous variants of unknown significance (c.128C > G [p.Pro43Arg]; c.776C > A [p.Pro259Gln]) were identified in LHX2 among the 70 patients. These variations were not identified in a panel of 100 control patients of mixed origins. The variation c.776C > A (p.Pro259Gln) was considered as non pathogenic by in silico analysis, while the variation c.128C > G (p.Pro43Arg) considered as deleterious by in silico analysis and was inherited from the asymptomatic father. Conclusions: Mutations in LHX2 do not represent a frequent cause of micro/anophthalmia.

FAM111A Mutations Result in Hypoparathyroidism and Impaired Skeletal Development.

Kenny-Caffey syndrome (KCS) and the similar but more severe osteocraniostenosis (OCS) are genetic conditions characterized by impaired skeletal development with small and dense bones, short stature, and primary hypoparathyroidism with hypocalcemia. We studied five individuals with KCS and five with OCS and found that all of them had heterozygous mutations in FAM111A. One mutation was identified in four unrelated individuals with KCS, and another one was identified in two unrelated individuals with OCS; all occurred de novo. Thus, OCS and KCS are allelic disorders of different severity. FAM111A codes for a 611 amino acid protein with homology to trypsin-like peptidases. Although FAM111A has been found to bind to the large T-antigen of SV40 and restrict viral replication, its native function is unknown. Molecular modeling of FAM111A shows that residues affected by KCS and OCS mutations do not map close to the active site but are clustered on a segment of the protein and are at, or close to, its outer surface, suggesting that the pathogenesis involves the interaction with as yet unidentified partner proteins rather than impaired catalysis. FAM111A appears to be crucial to a pathway that governs parathyroid hormone production, calcium homeostasis, and skeletal development and growth.

Novel FGF8 mutations associated with recessive holoprosencephaly, craniofacial defects, and hypothalamo-pituitary dysfunction.

Context: Fibroblast growth factor (FGF) 8 is important for GnRH neuronal development with human mutations resulting in Kallmann syndrome. Murine data suggest a role for Fgf8 in hypothalamo-pituitary development; however, its role in the etiology of wider hypothalamo-pituitary dysfunction in humans is unknown.Objective: The objective of this study was to screen for FGF8 mutations in patients with septo-optic dysplasia (n = 374) or holoprosencephaly (HPE)/midline clefts (n = 47).Methods: FGF8 was analyzed by PCR and direct sequencing. Ethnically matched controls were then screened for mutated alleles (n = 480-686). Localization of Fgf8/FGF8 expression was analyzed by in situ hybridization in developing murine and human embryos. Finally, Fgf8 hypomorphic mice (Fgf8(loxPNeo/-)) were analyzed for the presence of forebrain and hypothalamo-pituitary defects.Results: A homozygous p.R189H mutation was identified in a female patient of consanguineous parentage with semilobar HPE, diabetes insipidus, and TSH and ACTH insufficiency. Second, a heterozygous p.Q216E mutation was identified in a female patient with an absent corpus callosum, hypoplastic optic nerves, and Moebius syndrome. FGF8 was expressed in the ventral diencephalon and anterior commissural plate but not in Rathke's pouch, strongly suggesting early onset hypothalamic and corpus callosal defects in these patients. This was consolidated by significantly reduced vasopressin and oxytocin staining neurons in the hypothalamus of Fgf8 hypomorphic mice compared with controls along with variable hypothalamo-pituitary defects and HPE.Conclusion: We implicate FGF8 in the etiology of recessive HPE and potentially septo-optic dysplasia/Moebius syndrome for the first time to our knowledge. Furthermore, FGF8 is important for the development of the ventral diencephalon, hypothalamus, and pituitary. (J Clin Endocrinol Metab 96: E1709-E1718, 2011)

Mutations in the TGFβ binding-protein-like domain 5 of FBN1 are responsible for acromicric and geleophysic dysplasias.

Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although AD has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. After exome sequencing in GD and AD cases, we selected fibrillin 1 (FBN1) as a candidate gene, even though mutations in this gene have been described in Marfan syndrome, which is characterized by tall stature and arachnodactyly. We identified 16 heterozygous FBN1 mutations that are all located in exons 41 and 42 and encode TGFβ-binding protein-like domain 5 (TB5) of FBN1 in 29 GD and AD cases. Microfibrillar network disorganization and enhanced TGFβ signaling were consistent features in GD and AD fibroblasts. Importantly, a direct interaction between ADAMTSL2 and FBN1 was demonstrated, suggesting a disruption of this interaction as the underlying mechanism of GD and AD phenotypes. Although enhanced TGFβ signaling caused by FBN1 mutations can trigger either Marfan syndrome or GD and AD, our findings support the fact that TB5 mutations in FBN1 are responsible for short stature phenotypes.

Disease-associated mutations in the actin-binding domain of filamin B cause cytoplasmic focal accumulations correlating with disease severity.

Dominant missense mutations in FLNB, encoding the actin-cross linking protein filamin B (FLNB), cause a broad range of skeletal dysplasias with varying severity by an unknown mechanism. Here these FLNB mutations are shown to cluster in exons encoding the actin-binding domain (ABD) and filamin repeats surrounding the flexible hinge 1 region of the FLNB rod domain. Despite being positioned in domains that bind actin, it is unknown if these mutations perturb cytoskeletal structure. Expression of several full-length FLNB constructs containing ABD mutations resulted in the appearance of actin-containing cytoplasmic focal accumulations of the substituted protein to a degree that was correlated with the severity of the associated phenotypes. In contrast, study of mutations leading to substitutions in the FLNB rod domain that result in the same phenotypes as ABD mutations demonstrated that with only one exception disease-associated substitutions, surrounding hinge 1 demonstrated no tendency to form actin-filamin foci. The exception, a substitution in filamin repeat 6, lies within a region previously implicated in filamin-actin binding. These data are consistent with mutations in the ABD conferring enhanced actin-binding activity but suggest that substitutions affecting repeats near the flexible hinge region of FLNB precipitate the same phenotypes through a different mechanism.

Missense mutations in TENM4, a regulator of axon guidance and central myelination, cause essential tremor.

Essential tremor (ET) is a common movement disorder with an estimated prevalence of 5% of the population aged over 65 years. In spite of intensive efforts, the genetic architecture of ET remains unknown. We used a combination of whole-exome sequencing and targeted resequencing in three ET families. In vitro and in vivo experiments in oligodendrocyte precursor cells and zebrafish were performed to test our findings. Whole-exome sequencing revealed a missense mutation in TENM4 segregating in an autosomal-dominant fashion in an ET family. Subsequent targeted resequencing of TENM4 led to the discovery of two novel missense mutations. Not only did these two mutations segregate with ET in two additional families, but we also observed significant over transmission of pathogenic TENM4 alleles across the three families. Consistent with a dominant mode of inheritance, in vitro analysis in oligodendrocyte precursor cells showed that mutant proteins mislocalize. Finally, expression of human mRNA harboring any of three patient mutations in zebrafish embryos induced defects in axon guidance, confirming a dominant-negative mode of action for these mutations. Our genetic and functional data, which is corroborated by the existence of a Tenm4 knockout mouse displaying an ET phenotype, implicates TENM4 in ET. Together with previous studies of TENM4 in model organisms, our studies intimate that processes regulating myelination in the central nervous system and axon guidance might be significant contributors to the genetic burden of this disorder.

Galanin pathogenic mutations in temporal lobe epilepsy.

Temporal lobe epilepsy (TLE) is a common epilepsy syndrome with a complex etiology. Despite evidence for the participation of genetic factors, the genetic basis of TLE remains largely unknown. A role for the galanin neuropeptide in the regulation of epileptic seizures has been established in animal models more than two decades ago. However, until now there was no report of pathogenic mutations in GAL, the galanin-encoding gene, and therefore its role in human epilepsy was not established. Here, we studied a family with a pair of monozygotic twins affected by TLE and two unaffected siblings born to healthy parents. Exome sequencing revealed that both twins carried a novel de novo mutation (p.A39E) in the GAL gene. Functional analysis revealed that the p.A39E mutant showed antagonistic activity against galanin receptor 1 (GalR1)-mediated response, and decreased binding affinity and reduced agonist properties for GalR2. These findings suggest that the p.A39E mutant could impair galanin signaling in the hippocampus, leading to increased glutamatergic excitation and ultimately to TLE. In a cohort of 582 cases, we did not observe any pathogenic mutations indicating that mutations in GAL are a rare cause of TLE. The identification of a novel de novo mutation in a biologically-relevant candidate gene, coupled with functional evidence that the mutant protein disrupts galanin signaling, strongly supports GAL as the causal gene for the TLE in this family. Given the availability of galanin agonists which inhibit seizures, our findings could potentially have direct implications for the development of anti-epileptic treatment.

ERBB4 mutations in cancer and amyotrophic lateral sclerosis

ErbB receptor tyrosine kinases, epidermal growth factor receptor (EGFR, also known as ErbB1), ErbB2 (HER2 or NEU), ErbB3 (HER3), and ErbB4 (HER4), transduce signals borne by extracellular ligands into central cellular responses such as proliferation, survival, differentiation, and apoptosis. Mutations in ERBB genes are frequently detected in human malignant diseases of epithelial and neural origin, making ErbB receptors important drug targets. Targeting EGFR and ErbB2 has been successful in eg. lung and breast cancer, respectively, and mutations in these genes can be used to select patients that are responsive to the targeted treatment. Although somatic ERBB4 mutations have been found in many high-incidence cancers such as melanoma, lung cancer, and colorectal cancer and germ-line ERBB4 mutations have been linked to neuronal disorders and cancer, ErbB4 has generally been neglected as a potential drug target. Thus, the consequences of ERBB4 mutations on ErbB4 biology are largely unknown. This thesis aimed to elucidate the functional consequences and assess the clinical significance of somatic and germ-line ERBB4 mutations in the context of cancer and amyotrophic lateral sclerosis. The results of this study indicated that cancer-associated ERBB4 mutations can promote aberrant ErbB4 function by activating the receptor or inducing qualitative changes in ErbB4 signaling. ERBB4 mutations increased survival or decreased differentiation in vitro, suggesting that ERBB4 mutations can be oncogenic. Importantly, the potentially oncogenic mutations were located in various subdomains in ErbB4, possibly providing explanation for the characteristic scattered pattern of mutations in ERBB4. This study also demonstrated that hereditary variation in ERBB4 gene can have a significant effect on the prognosis of breast cancer. In addition, it was shown that hereditary or de novo germ-line ERBB4 mutations that predispose to amyotrophic lateral sclerosis inhibit ErbB4 activity. Together, these results suggest that ErbB4 should be considered as a novel drug target in cancer and amyotrophic lateral sclerosis.

Prevalence of GJB2 (Connexin-26) and GJB6 (Connexin-30) Mutations in a Cohort of 300 Brazilian Hearing-Impaired Individuals: Implications for Diagnosis and Genetic Counseling

Objective: Hereditary nonsyndromic deafness is an autosomal recessive condition in about 80% of cases, and point mutations in the GJB2 gene (connexin 26) and two deletions in the GJB6 gene (connexin 30), del(GJB6-D13S1830) and del(GJB6-D13S1854), are reported to account for 50% of recessive deafness, Aiming at establishing the frequencies of GJB2 mutations and GJB6 deletions in the Brazilian population, we screened 300 unrelated individuals with hearing impairment, who were not affected by known deafness related syndromes. Methods: We firstly screened the most frequently reported mutations, c.35delG and c.167delT in the GJB2 gene, and del(GJB6-D13S1830) and del(GJB6-D13S1854) in the GJB6 gene, through specific techniques. The detected c.35delG and c.167delT mutations were validated by sequencing. Other mutations in the GJB2 gene were screened by single-strand conformation polymorphism and the coding region was sequenced when abnormal patterns were found. Results: Pathogenic mutations in GJB2 and GJB6 genes were detected in 41 individuals (13.7%), and 80.5% (33/41) presented these mutations in homozygosis or compound heterozygosis, thus explaining their hearing defect. The c.35delG in the GJB2 gene was the most frequent mutation (37/300; 12.4%), detected in 23% familial and 6.2% the sporadic cases. The second most frequent mutation (1%; 3/300) was the del(GJB6- D13S1830), always found associated with the c.35delG mutation. Nineteen different sequence variations were found in the GJB2 gene. In addition to the c.35delG mutation, nine known pathogenic alterations were detected 0 67delT, p.Trp24X, p.Val37lle, c.176_191del16, c.235delC, p.Leu90Pro, p.Arg127His, c.509insA, and p.Arg184Pro, Five substitutions had been previously considered benign polymorphisms: c.-15C>T, p.Val27lle, p.Met34hr, p.Ala40Ala, and p.Gly160Ser. Two previously reported Mutations of unknown pathogenicity were found (p.Lys168Arg, and c.684C>A), and two novel substitutions, p.Leu81Val (c.G241C) and p.Met195Val (c.A583G), both in heterozygosis without an accompanying mutation in the other allele. None of these latter four variants of undefined status was present in a sample of 100 hearing controls. Conclusions: The present study demonstrates that Mutations in the GJB2 gene and del(GJB6 D13S1830) are important causes of hearing impairment in Brazil, thus justifying their screening in a routine basis. The diversity of variants in our sample reflects the ethnic heterogeneity of the Brazilian population.

WNT5A Mutations in Patients With Autosomal Dominant Robinow Syndrome

Robinow syndrome is a skeletal dysplasia with both autosomal dominant and autosomal recessive inheritance patterns. It is characterized by short stature, limb shortening, genital hypoplasia, and craniofacial abnormalities. The etiology of dominant Robinow syndrome is unknown; however, the phenotypically more severe autosomal recessive form of Robinow syndrome has been associated with mutations in the orphan tyrosine kinase receptor, ROR2, which has recently been identified as a putative WNT5A receptor. Here, we show that two different missense mutations in WNT5A, which result in amino acid substitutions of highly conserved cysteines, are associated with autosomal dominant Robinow syndrome. One mutation has been found in all living affected members of the original family described by Meinhard Robinow and another in a second unrelated patient. These missense mutations result in decreased WNT5A activity in functional assays of zebrafish and Xenopus development. This work suggests that a WNT5A/ROR2 signal transduction pathway is important in human craniofacial and skeletal development and that proper formation and growth of these structures is sensitive to variations in WNT5A function. Developmental Dynamics 239:327-337, 2010. (C) 2009 Wiley-Liss, Inc.

Mutations in IRF6 cause Van der Woude and popliteal pterygium syndromes

Interferon regulatory factor 6 (IRF6) belongs to a family of nine transcription factors that share a highly conserved helix-turn-helix DNA-binding domain and a less conserved protein-binding domain. Most IRFs regulate the expression of interferon-alpha and -beta after viral infection(1), but the function of IRF6 is unknown. The gene encoding IRF6 is located in the critical region for the Van der Woude syndrome (VWS; OMIM 119300) locus at chromosome 1q32-q41 (refs 2,3). The disorder is an autosomal dominant form of cleft lip and palate with lip pits(4), and is the most common syndromic form of cleft lip or palate. Popliteal pterygium syndrome (PPS; OMIM 119500) is a disorder with a similar orofacial phenotype that also includes skin and genital anomalies(5). Phenotypic overlap(6) and linkage data(7) suggest that these two disorders are allelic. We found a nonsense mutation in IRF6 in the affected twin of a pair of monozygotic twins who were discordant for VWS. Subsequently, we identified mutations in IRF6 in 45 additional unrelated families affected with VWS and distinct mutations in 13 families affected with PPS. Expression analyses showed high levels of Irf6 mRNA along the medial edge of the fusing palate, tooth buds, hair follicles, genitalia and skin. Our observations demonstrate that haploinsufficiency of IRF6 disrupts orofacial development and are consistent with dominant-negative mutations disturbing development of the skin and genitalia.

Expanding the spectrum of PTH1R mutations in patients with primary failure of tooth eruption

Objectives: Primary failure of tooth eruption (PFE) is a rare autosomal-dominant disease characterized by severe lateral open bite as a consequence of incomplete eruption of posterior teeth. Heterozygous mutations in the parathyroid hormone 1 receptor (PTH1R) gene have been shown to cause PFE likely due to protein haploinsufficiency. To further expand on the mutational spectrum of PFE-associated mutations, we report here on the sequencing results of the PTH1R gene in 70 index PFE cases. Materials and methods: Sanger sequencing of the PTH1R coding exons and their immediate flanking intronic sequences was performed with DNA samples from 70 index PFE cases. Results: We identified a total of 30 unique variants, of which 12 were classified as pathogenic based on their deleterious consequences on PTH1R protein while 16 changes were characterized as unclassified variants with as yet unknown effects on disease pathology. The remaining two variants represent common polymorphisms. Conclusions: Our data significantly increase the number of presently known unique PFE-causing PTH1R mutations and provide a series of variants with unclear pathogenicity which will require further in vitro assaying to determine their effects on protein structure and function. Clinical relevance: Management of PTH1R-associated PFE is problematic, in particular when teeth are exposed to orthodontic force. Therefore, upon clinical suspicion of PFE, molecular DNA testing is indicated to support decision making for further treatment options. © 2013 Springer-Verlag Berlin Heidelberg.

Detailed analyses of mutations affecting structural formation of neuromuscular synapses in Drosophila melanogaster

The establishment of appropriate synapses between neurons and their target cells is an essential requirement for the formation of functional neuronal circuits. However, there is very little insight into the mechanisms underlying de novo formation of synapses and synaptic terminals. To identify novel genes involved in signalling or structural aspects of these processes I capitalised on possibilities provided by the model organism Drosophila. Thus, I contributed to a screen of a collection of third chromosomal mutations (Salzberg et al., 1997, Genetics 147, 1723ff.) selecting those mutant strains displaying structural defects of Drosophila neuromuscular junctions (NMJ). Carrying out genetic mapping experiments, I could assign 7 genes to interesting candidate mutations. All 7 mutations selected in this process cause size alterations of the embryonic NMJ, and one shows additional disturbances in the distribution of synaptic markers. 4 of these turned out to be transcription factors, not falling into the remit of this project. Only for one of these, the neuronal transcription factor Castor, I could show that its overgrown mutant NMJ phenotype is due to an increase in the number of motorneurons. The remaining genes encode a potential nitrophenylphosphatase, the translation initiation factor eIF4AIII, and a novel protein Waharan. Unfortunately, the nitophenylphosphatase gene was identified too late to carry out functional studies in the context of this project, but potential roles are discussed. eIF4AIII promotes NMJ size tempting to speculate that local translation at the NMJ is affected. I found that the synaptic scaffolding molecule Discs large (Dlg; orthologue of PSD95) is upregulated at eIF4AIII mutant NMJs. Targeted upregulation of Dlg can not mimic the eIF4AIII mutant phenotype, but dlg mutations suppress it. Therefore, Dlg function is required but not sufficient in this context. My findings are discussed in detail, pointing out future directions. The main focus of this work is the completely novel gene waharan (wah), an orthologue of the human gene KIAA1267 encoding a big brain protein of likewise unknown structure and function. My studies show that mutations or RNAi knock-down of wah cause NMJ overgrowth and reveal additional crucial roles in the patterning of wing imginal discs. RNAi studies suggest Wah to be required pre- and postsynaptically at NMJs and, consistently, wah is transcribed in the nervous system and muscles. Anti-Wah antisera were produced but could no longer be tested here, but preliminary studies with newly generated HA-targeted constructs suggest that Wah localises at NMJs and in neuronal nuclei. In silico analyses predict Wah to be structurally related to the Rad23-family of proteins, likely to target ubiquitinated proteins to the proteasome for degradation (Chen et al., 2002, Mol Cell Biol 22, 4902ff.) . In agreement with this prediction, poly-ubiquitinated proteins were found to accumulate in the absence of wah function, and wah-like mutant phenotypes were induced in NMJs and wing discs by knocking down proteasome function. My analysis further revealed that poly-ubiquitinated proteins are reduced in nuclei of wah mutant neurons and muscles, suggesting that Wah may play additional roles in ubiquitin-mediated nuclear import. Taken together, this study has uncovered a number of interesting candidate genes required for the de novo formation of Drosophila NMJs. 3 of these genes fell into the focus of this project. As discussed in detail, discovery of these genes and insights gained into their function have high potential to be translatable into vertebrate systems.

Detecting BRAF Mutations in Formalin-Fixed Melanoma: Experiences with Two State-of-the-Art Techniques

Melanoma is characterized by a high frequency of BRAF mutations. It is unknown if the BRAF mutation status has any predictive value for therapeutic approaches such as angiogenesis inhibition.

Novel mutations consolidate KCTD7 as a progressive myoclonus epilepsy gene

The progressive myoclonus epilepsies (PMEs) comprise a group of clinically and genetically heterogeneous disorders characterised by myoclonus, epilepsy, and neurological deterioration. This study aimed to identify the underlying gene(s) in childhood onset PME patients with unknown molecular genetic background.