Kerstin Kutsche

Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D.

Usher syndrome type I (USH1) is an autosomal recessive disorder characterized by congenital sensorineural hearing loss, vestibular dysfunction and visual impairment due to early onset retinitis pigmentosa1 (RP). So far, six loci (USH1A–USH1F) have been mapped, but only two USH1 genes have been identified: MYO7A (ref. 2) for USH1B and the gene encoding harmonin3,4 for USH1C. We identified a Cuban pedigree linked to the locus for Usher syndrome type 1D (MIM 601067) within the q2 region of chromosome 10 (ref. 5). Affected individuals present with congenital deafness and a highly variable degree of retinal degeneration. Using a positional candidate approach, we identified a new member of the cadherin gene superfamily, CDH23. It encodes a protein of 3,354 amino acids with a single transmembrane domain and 27 cadherin repeats. In the Cuban family, we detected two different mutations: a severe course of the retinal disease was observed in individuals homozygous for what is probably a truncating splice-site mutation (c.4488G→C), whereas mild RP is present in individuals carrying the homozygous missense mutation R1746Q. A variable expression of the retinal phenotype was seen in patients with a combination of both mutations. In addition, we identified two mutations, ΔM1281 and IVS51+5G→A, in a German USH1 patient. Our data show that different mutations in CDH23 result in USH1D with a variable retinal phenotype. In an accompanying paper6, it is shown that mutations in the mouse ortholog cause disorganization of inner ear stereocilia and deafness in the waltzer mouse.

Mutations in ARHGEF6, encoding a guanine nucleotide exchange factor for Rho GTPases, in patients with X-linked mental retardation.

X-linked forms of mental retardation (XLMR) include a variety of different disorders and may account for up to 25% of all inherited cases of mental retardation. So far, seven X-chromosomal genes mutated in nonspecific mental retardation (MRX) have been identified: FMR2, GDI1, RPS6KA3, IL1RAPL, TM4SF2, OPHN1 and PAK3 (refs 2–9). The products of the latter two have been implicated in regulation of neural plasticity by controlling the activity of small GTPases of the Rho family. Here we report the identification of a new MRX gene, ARHGEF6 (also known as αPIX or Cool-2), encoding a protein with homology to guanine nucleotide exchange factors for Rho GTPases (Rho GEF). Molecular analysis of a reciprocal X/21 translocation in a male with mental retardation showed that this gene in Xq26 was disrupted by the rearrangement. Mutation screening of 119 patients with nonspecific mental retardation revealed a mutation in the first intron of ARHGEF6 (IVS1-11T→C) in all affected males in a large Dutch family. The mutation resulted in preferential skipping of exon 2, predicting a protein lacking 28 amino acids. ARHGEF6 is the eighth MRX gene identified so far and the third such gene to encode a protein that interacts with Rho GTPases.

Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes

N-methyl-D-aspartate (NMDA) receptors mediate excitatory neurotransmission in the mammalian brain. Two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits each form highly Ca2+-permeable cation channels which are blocked by extracellular Mg2+ in a voltage-dependent manner. Either GRIN2B or GRIN2A, encoding the NMDA receptor subunits NR2B and NR2A, was found to be disrupted by chromosome translocation breakpoints in individuals with mental retardation and/or epilepsy. Sequencing of GRIN2B in 468 individuals with mental retardation revealed four de novo mutations: a frameshift, a missense and two splice-site mutations. In another cohort of 127 individuals with idiopathic epilepsy and/or mental retardation, we discovered a GRIN2A nonsense mutation in a three-generation family. In a girl with early-onset epileptic encephalopathy, we identified the de novo GRIN2A mutation c.1845C>A predicting the amino acid substitution p.N615K. Analysis of NR1-NR2AN615K (NR2A subunit with the p.N615K alteration) receptor currents revealed a loss of the Mg2+ block and a decrease in Ca2+ permeability. Our findings suggest that disturbances in the neuronal electrophysiological balance during development result in variable neurological phenotypes depending on which NR2 subunit of NMDA receptors is affected.

Mutation of SHOC2 promotes aberrant protein N-myristoylation and causes Noonan-like syndrome with loose anagen hair

N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue. We show that aberrantly acquired N-myristoylation of SHOC2, a leucine-rich repeat–containing protein that positively modulates RAS-MAPK signal flow, underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair (MIM607721) shared the 4A>G missense change in SHOC2 (producing an S2G amino acid substitution) that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2S2G in vitro enhanced MAPK activation in a cell type–specific fashion. Induction of SHOC2S2G in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease.

A restricted spectrum of NRAS mutations causes Noonan syndrome

Noonan syndrome, a developmental disorder characterized by congenital heart defects, reduced growth, facial dysmorphism and variable cognitive deficits, is caused by constitutional dysregulation of the RAS-MAPK signaling pathway. Here we report that germline NRAS mutations conferring enhanced stimulus-dependent MAPK activation account for some cases of this disorder. These findings provide evidence for an obligate dependency on proper NRAS function in human development and growth.

Mutations of CASK cause an X-linked brain malformation phenotype with microcephaly and hypoplasia of the brainstem and cerebellum

CASK is a multi-domain scaffolding protein that interacts with the transcription factor TBR1 and regulates expression of genes involved in cortical development such as RELN. Here we describe a previously unreported X-linked brain malformation syndrome caused by mutations of CASK. All five affected individuals with CASK mutations had congenital or postnatal microcephaly, disproportionate brainstem and cerebellar hypoplasia, and severe mental retardation.CASK is a multi-domain scaffolding protein that interacts with the transcription factor TBR1 and regulates expression of genes involved in cortical development such as RELN. Here we describe a previously unreported X-linked brain malformation syndrome caused by mutations of CASK. All five affected individuals with CASK mutations had congenital or postnatal microcephaly, disproportionate brainstem and cerebellar hypoplasia, and severe mental retardation.

Expansion of the genotypic and phenotypic spectrum in patients with KRAS germline mutations

Background: Noonan syndrome, cardio-facio-cutaneous syndrome (CFC) and Costello syndrome constitute a group of developmental disorders with an overlapping pattern of congenital anomalies. Each of these conditions can be caused by germline mutations in key components of the highly conserved Ras-MAPK pathway, possibly reflecting a similar pathogenesis underlying the three disorders. Germline mutations in KRAS have recently been identified in a small number of patients with Noonan syndrome and CFC. Methods and results: 260 patients were screened for KRAS mutations by direct sequencing. Overall, we detected KRAS mutations in 12 patients, including three known and eight novel sequence alterations. All mutations are predicted to cause single amino acid substitutions. Remarkably, our cohort of individuals with KRAS mutations showed a high clinical variability, ranging from Noonan syndrome to CFC, and also included two patients who met the clinical criteria of Costello syndrome. Conclusion: Our findings reinforce the picture of a clustered distribution of disease associated KRAS germline alterations. We further defined the phenotypic spectrum associated with KRAS missense mutations and provided the first evidence of clinical differences in patients with KRAS mutations compared with Noonan syndrome affected individuals with heterozygous PTPN11 mutations and CFC patients carrying a BRAF, MEK1 or MEK1 alteration, respectively. We speculate that the observed phenotypic variability may be related, at least in part, to specific genotypes and possibly reflects the central role of K-Ras in a number of different signalling pathways.

The core FOXG1 syndrome phenotype consists of postnatal microcephaly, severe mental retardation, absent language, dyskinesia, and corpus callosum hypogenesis

Background Submicroscopic deletions in 14q12 spanning FOXG1 or intragenic mutations have been reported in patients with a developmental disorder described as a congenital variant of Rett syndrome. This study aimed to further characterise and delineate the phenotype of FOXG1 mutation positive patients. Method The study mapped the breakpoints of a 2;14 translocation by fluorescence in situ hybridisation and analysed three chromosome rearrangements in 14q12 by cytogenetic analysis and/or array comparative genomic hybridisation. The FOXG1 gene was sequenced in 210 patients, including 129 patients with unexplained developmental disorders and 81 MECP2 mutation negative individuals. Results One known mutation, seen in two patients, and nine novel mutations of FOXG1 including two deletions, two chromosome rearrangements disrupting or displacing putative cis-regulatory elements from FOXG1, and seven sequence changes, are reported. Analysis of 11 patients in this study, and a further 15 patients reported in the literature, demonstrates a complex constellation of features including mild postnatal growth deficiency, severe postnatal microcephaly, severe mental retardation with absent language development, deficient social reciprocity resembling autism, combined stereotypies and frank dyskinesias, epilepsy, poor sleep patterns, irritability in infancy, unexplained episodes of crying, recurrent aspiration, and gastro-oesophageal reflux. Brain imaging studies reveal simplified gyral pattern and reduced white matter volume in the frontal lobes, corpus callosum hypogenesis, and variable mild frontal pachgyria. Conclusions These findings have significantly expanded the number of FOXG1 mutations and identified two affecting possible cis-regulatory elements. While the phenotype of the patients overlaps both classic and congenital Rett syndrome, extensive clinical evaluation demonstrates a distinctive and clinically recognisable phenotype which the authors suggest designating as the FOXG1 syndrome.

SOS1 is the second most common Noonan gene but plays no major role in cardio-facio-cutaneous syndrome

Background: Heterozygous gain-of-function mutations in various genes encoding proteins of the Ras-MAPK signalling cascade have been identified as the genetic basis of Noonan syndrome (NS) and cardio-facio-cutaneous syndrome (CFCS). Mutations of SOS1, the gene encoding a guanine nucleotide exchange factor for Ras, have been the most recent discoveries in patients with NS, but this gene has not been studied in patients with CFCS. Methods and results: We investigated SOS1 in a large cohort of patients with disorders of the NS–CFCS spectrum, who had previously tested negative for mutations in PTPN11, KRAS, BRAF, MEK1 and MEK2. Missense mutations of SOS1 were discovered in 28% of patients with NS. In contrast, none of the patients classified as having CFCS was found to carry a pathogenic sequence change in this gene. Conclusion: We have confirmed SOS1 as the second major gene for NS. Patients carrying mutations in this gene have a distinctive phenotype with frequent ectodermal anomalies such as keratosis pilaris and curly hair. However, the clinical picture associated with SOS1 mutations is different from that of CFCS. These findings corroborate that, despite being caused by gain-of-function mutations in molecules belonging to the same pathway, NS and CFCS scarcely overlap genotypically.

Mutation and phenotypic spectrum in patients with cardio-facio-cutaneous and Costello syndrome.

Cardio‐facio‐cutaneous (CFC) and Costello syndrome (CS) are congenital disorders with a significant clinical overlap. The recent discovery of heterozygous mutations in genes encoding components of the RAS–RAF–MAPK pathway in both CFC and CS suggested a similar underlying pathogenesis of these two disorders. While CFC is heterogeneous with mutations in BRAF, MAP2K1, MAP2K2 and KRAS, HRAS alterations are almost exclusively associated with CS. We carried out a comprehensive mutation analysis in 51 CFC‐affected patients and 31 individuals with CS. Twelve different BRAF alterations were found in twenty‐four patients with CFC (47.0%), two MAP2K1 mutations in five (9.8%) and two MAP2K2 sequence variations in three CFC‐affected individuals (5.9%), whereas three patients had a KRAS alteration (5.9%). We identified four different missense mutations of HRAS in twenty‐eight cases with CS (90.3%), while KRAS mutations were detected in two infants with a phenotype meeting criteria for CS (6.5%). In 14 informative families, we traced the parental origin of HRAS alterations and demonstrated inheritance of the mutated allele exclusively from the father, further confirming a paternal bias in the parental origin of HRAS mutations in CS. Careful clinical evaluation of patients with BRAF and MAP2K1/2 alterations revealed the presence of slight phenotypic differences regarding craniofacial features in MAP2K1‐ and MAP2K2‐mutation positive individuals, suggesting possible genotype–phenotype correlations.