Missense mutations in the most ancient residues of the PAX6 paired domain underlie a spectrum of human congenital eye malformations

Mutations of the human PAX6 gene underlie aniridia (congenital absence of the iris), a rare dominant malformation of the eye. The spectrum of PAX6 mutations in aniridia patients is highly biased, with 92% of all reported mutations leading to premature truncation of the protein (nonsense, splicing, insertions and deletions) and just 2% leading to substitution of one amino acid by another (missense). The extraordinary conservation of the PAX6 protein at the amino acid level amongst vertebrates predicts that pathological missense mutations should in fact be common even though they are hardly ever seen in aniridia patients. This indicates that there is a heavy ascertainment bias in the selection of patients for PAX6 mutation analysis and that the 'missing' PAX6 missense mutations frequently may underlie phenotypes distinct from textbook aniridia. Here we present four novel PAX6 missense mutations, two in association with atypical phenotypes: ectopia pupillae (displaced pupils) and congenital nystagmus (searching gaze), and two in association with more recognizable aniridia phenotypes. Strikingly, all four mutations are located within the PAX6 paired domain and affect amino acids which are highly conserved in all known paired domain proteins. Our results support the hypothesis that the under-representation of missense mutations is caused by ascertainment bias and suggest that a substantial burden of PAX6 - related disease remains to be uncovered.      

Characterization of phenylketonuria missense substitutions, distant from the phenylalanine hydroxylase active site, illustrates a paradigm for mechanism and potential modulation of phenotype

Missense mutations account for 48% of all reported human disease-causing alleles. Since few are predicted to ablate directly an enzyme's catalytic site or other functionally important amino acid residues, how do most missense mutations cause loss of function and lead to disease? The classic monogenic phenotype hyperphenylalaninemia (HPA), manifesting notably as phenylketonuria (PKU), where missense mutations in the PAH gene compose 60% of the alleles impairing phenylalanine hydroxylase (PAH) function, allows us to examine this question. Here we characterize four PKU-associated PAH mutations (F39L, K42I, L48S, I65T), each changing an amino acid distant from the enzyme active site. Using three complementary in vitro protein expression systems, and 3D-structural localization, we demonstrate a common mechanism. PAH protein folding is affected, causing altered oligomerization and accelerated proteolytic degradation, leading to reduced cellular levels of this cytosolic protein. Enzyme specific activity and kinetic properties are not adversely affected, implying that the only way these mutations reduce enzyme activity within cells in vivo is by producing structural changes which provoke the cell to destroy the aberrant protein. The F39L, L48S, and I65T PAH mutations were selected because each is associated with a spectrum of in vivo HPA among patients. Our in vitro data suggest that interindividual differences in cellular handling of the mutant, but active, PAH proteins will contribute to the observed variability of phenotypic severity. PKU thus supports a newly emerging paradigm both for mechanism whereby missense mutations cause genetic disease and for potential modulation of a disease phenotype.     

Pathogenic or not? And if so,then how? Studying the effects of missense mutations using bioinformatics methods

Many gene defects are relatively easy to identify experimentally, but obtaining information about the effects of sequence variations and elucidation of the detailed molecular mechanisms of genetic diseases will be among the next major efforts in mutation research. Amino acid substitutions may have diverse effects on protein structure and function; thus, a detailed analysis of the mutations is essential. Experimental study of the molecular effects of mutations is laborious, whereas useful and reliable information about the effects of amino acid substitutions can readily be obtained by theoretical methods. Experimentally defined structures and molecular modeling can be used as a basis for interpretation of the mutations. The effects of missense mutations can be analyzed even when the 3D structure of the protein has not been determined, although structure‐based analyses are more reliable. Structural analyses include studies of the contacts between residues, their implication for the stability of the protein, and the effects of the introduced residues. Investigations of steric and stereochemical consequences of substitutions provide insights on the molecular fit of the introduced residue. Mutations that change the electrostatic surface potential of a protein have wide‐ranging effects. Analyses of the effects of mutations on interactions with ligands and partners have been performed for elucidation of functional mutations. We have employed numerous methods for predicting the effects of amino acid substitutions. We discuss the applicability of these methods in the analysis of genes, proteins, and diseases to reveal protein structure–function relationships, which is essential to gain insights into disease genotype–phenotype correlations. Hum Mutat 0, 1–15, 2009. © 2009 Wiley‐Liss, Inc.     

DCC mutation update: Congenital mirror movements,isolated agenesis of the corpus callosum,and developmental split brain syndrome

The deleted in colorectal cancer (DCC) gene encodes the netrin‐1 (NTN1) receptor DCC, a transmembrane protein required for the guidance of commissural axons. Germline DCC mutations disrupt the development of predominantly commissural tracts in the central nervous system (CNS) and cause a spectrum of neurological disorders. Monoallelic, missense, and predicted loss‐of‐function DCC mutations cause congenital mirror movements, isolated agenesis of the corpus callosum (ACC), or both. Biallelic, predicted loss‐of‐function DCC mutations cause developmental split brain syndrome (DSBS). Although the underlying molecular mechanisms leading to disease remain poorly understood, they are thought to stem from reduced or perturbed NTN1 signaling. Here, we review the 26 reported DCC mutations associated with abnormal CNS development in humans, including 14 missense and 12 predicted loss‐of‐function mutations, and discuss their associated clinical characteristics and diagnostic features. We provide an update on the observed genotype–phenotype relationships of congenital mirror movements, isolated ACC and DSBS, and correlate this to our current understanding of the biological function of DCC in the development of the CNS. All mutations and their associated phenotypes were deposited into a locus‐specific LOVD ( https://databases.lovd.nl/shared/genes/DCC ).     

A novel classification system to predict the pathogenic effects of CHD7 missense variants in CHARGE syndrome

CHARGE syndrome is characterized by the variable occurrence of multisensory impairment, congenital anomalies, and developmental delay, and is caused by heterozygous mutations in the CHD7 gene. Correct interpretation of CHD7 variants is essential for genetic counseling. This is particularly difficult for missense variants because most variants in the CHD7 gene are private and a functional assay is not yet available. We have therefore developed a novel classification system to predict the pathogenic effects of CHD7 missense variants that can be used in a diagnostic setting. Our classification system combines the results from two computational algorithms (PolyPhen-2 and Align-GVGD) and the prediction of a newly developed structural model of the chromo- and helicase domains of CHD7 with segregation and phenotypic data. The combination of different variables will lead to a more confident prediction of pathogenicity than was previously possible. We have used our system to classify 145 CHD7 missense variants. Our data show that pathogenic missense mutations are mainly present in the middle of the CHD7 gene, whereas benign variants are mainly clustered in the 5' and 3' regions. Finally, we show that CHD7 missense mutations are, in general, associated with a milder phenotype than truncating mutations.     

CYP21 mutations in simple virilizing congenital adrenal hyperplasia

We studied the functional and structural effects of two unique missense mutations in CYP21 found in patients with simple virilizing congenital adrenal hyperplasia. The rare variants L300F and V281G were found in two girls who were each hemizygous for one of the mutations. Functional analysis after expression in COS-1 cells revealed that the mutant enzymes had reduced enzymatic activity for conversion of both 17-hydroxyprogesterone (L300F 9.5%, V281G 3.9% of normal) and progesterone (L300F 4.4%, V281G 3.9% of normal). Both mutant enzymes had an increased degradation in mammalian COS-1 cells compared to the normal protein, although the L300F variant affected the degradation pattern to a greater extent. Our data indicate that the residue L300 is important in maintaining normal structure of the 21-hydroxylase enzyme whereas mutations affecting V281 most likely cause impaired enzyme activity by interfering with a specific function(s) of the protein.     

MUSK, a new target for mutations causing congenital myasthenic syndrome

We report the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed dramatic pre- and postsynaptic structural abnormalities of the neuromuscular junction and severe decrease in acetylcholine receptor (AChR) epsilon-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The frameshift mutation led to the absence of MuSK expression. The missense mutation did not affect MuSK catalytic kinase activity but diminished expression and stability of MuSK leading to decreased agrin-dependent AChR aggregation, a critical step in the formation of the neuromuscular junction. In electroporated mouse muscle, overexpression of the missense mutation induced, within a week, a phenotype similar to the patient muscle biopsy: a severe decrease in synaptic AChR and an aberrant axonal outgrowth. These results strongly suggest that the missense mutation, in the presence of a null mutation on the other allele, is responsible for the dramatic synaptic changes observed in the patient.     

Genetic Insights into Congenital Neutropenia

Congenital neutropenia syndromes comprise a heterogeneous group of disorders leading to increased susceptibility to bacterial infections. Recent work has elucidated the molecular basis of several congenital neutropenia syndromes such as mutations in ELA2, HAX1, GF11, and WAS. In addition, a number of complex clinical syndromes associating congenital neutropenia have been recognized and elucidated on a genetic level, e.g. p14-deficiency or G6PC3-deficiency. The clinical and genetic findings of various neutropenia syndromes are being discussed.     

R91W mutation in Rpe65 leads to milder early-onset retinal dystrophy due to the generation of low levels of 11-cis-retinal

RPE65 is a retinal pigment epithelial protein essential for the regeneration of 11-cis-retinal, the chromophore of cone and rod visual pigments. Mutations in RPE65 lead to a spectrum of retinal dystrophies ranging from Leber's congenital amaurosis to autosomal recessive retinitis pigmentosa. One of the most frequent missense mutations is an amino acid substitution at position 91 (R91W). Affected patients have useful cone vision in the first decade of life, but progressively lose sight during adolescence. We generated R91W knock-in mice to understand the mechanism of retinal degeneration caused by this aberrant Rpe65 variant. We found that in contrast to Rpe65 null mice, low but substantial levels of both RPE65 and 11-cis-retinal were present. Whereas rod function was impaired already in young animals, cone function was less affected. Rhodopsin metabolism and photoreceptor morphology were disturbed, leading to a progressive loss of photoreceptor cells and retinal function. Thus, the consequences of the R91W mutation are clearly distinguishable from an Rpe65 null mutation as evidenced by the production of 11-cis-retinal and rhodopsin as well as by less severe morphological and functional disturbances at early age. Taken together, the pathology in R91W knock-in mice mimics many aspects of the corresponding human blinding disease. Therefore, this mouse mutant provides a valuable animal model to test therapeutic concepts for patients affected by RPE65 missense mutations.     

EGFR regulates RhoA-GTP dependent cell motility in E-cadherin mutant cells

Gastric cancer associated E-cadherin germline missense mutations lead to significant functional consequences, in both the structural and signalling properties of the protein. In this study, we have characterized the effect of four E-cadherin germline missense mutations (T340A, A634V, P799R and V832M) in the interaction with the epidermal growth factor receptor (EGFR). We challenged the hypothesis that E-cadherin mutations perturb its ability to bind to EGFR, leading to constitutional activation of the EGFR, triggering activation of downstream effectors. We verified that missense mutations localized in the extracellular domain of the protein (T340A and A634V) exhibited reduced stability of the EGFR/E-cadherin heterodimers in contrast to germline mutations localized at the cytoplasmatic domain of the protein (P799R and V832M). We observed that cells expressing E-cadherin extracellular mutants displayed increased levels of phosphorylated EGFR upon ligand stimulation, when compared with cells expressing wild-type E-cadherin or intracellular mutants. We showed that upon treatment of E-cadherin extracellular mutant cells with the EGFR inhibitor, the increase of RhoA activation is abrogated and accompanied by decreased migratory behaviour, supporting the idea that Rho-like proteins are EGFR downstream effectors. Our results bring new insights into the understanding of the distinct in vitro behaviours observed for E-cadherin missense mutations localized in different domains of the protein. Furthermore, we demonstrate that E-cadherin-dependent EGFR activation contributes to enhanced cell motility, in a mechanism involving RhoA activation.     

Novel missense mutations of TMPRSS3 in two consanguineous Tunisian families with non‐syndromic autosomal recessive deafness

Recently the TMPRSS3 gene, which encodes a transmembrane serine protease, was found to be responsible for two non‐syndromic recessive deafness loci located on human chromosome 21q22.3, DFNB8 and DFNB10. We found evidence for linkage to the DFNB8/10 locus in two unrelated consanguineous Tunisian families segregating congenital autosomal recessive sensorineural deafness. The audiometric tests showed a loss of hearing greater than 70 dB, in all affected individuals of both families. Mutation screening of TMPRSS3 revealed two novel missense mutations, W251C and P404L, altering highly conserved amino acids of the serine protease domain. Both mutations were not found in 200 control Tunisian chromosomes. The detection of naturally‐occurring TMPRSS3 missense mutations in deafness families identifies functionally important amino acids. Comparative protein modeling of the TMPRSS3 protease domain predicted that W251C might lead to a structural rearrangement affecting the active site H257 and that P404L might alter the geometry of the active site loop and therefore affect the serine protease activity. Hum Mutat 18:101–108, 2001. © 2001 Wiley‐Liss, Inc.     

Molecular basis of multiple exostoses: mutations in the EXT1 and EXT2 genes

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the formation of exostoses, which are cartilage-capped bony protuberances mainly located on long bones. Two genes, EXT1 and EXT2, and at least one other unidentified gene, are known to be involved in the formation of exostoses. To date, 49 different EXT1 and 25 different EXT2 mutations have been found in EXT patients, and there is evidence that mutations in these two genes are responsible for over 70% of the EXT cases. Among the 49 EXT1 mutations there are 9 nonsense, 21 frameshift, and 5 splice site mutations; 2 in-frame deletions of 1 and 5 amino acids respectively; and 12 missense mutations. For EXT2, 8 nonsense, 11 frameshift, 3 splice site and 3 missense mutations are described. The majority of these mutations are mutations causing loss of function, which is consistent with the presumed tumor suppressor function of the EXT genes.     

Novel mutations in the human sucrase-isomaltase gene (SI) that cause congenital carbohydrate malabsorption

Disaccharide intolerance I or congenital sucrase-isomaltase deficiency (CSID) is a disorder leading to maldigestion of disaccharides, which is autosomal recessively inherited. Here we analyzed the sucrase-isomaltase (SI) gene from 11 patients of Hungarian origin with congenital sucrase-isomaltase deficiency. Variants in the SI gene had previously been described in CSID patients, which cause amino acid exchanges that affect the transport, the processing, or the function of the SI protein. None of our patients had known mutations for CSID. Our analyses revealed 43 SI variants in total, 15 within exons and one at a splice site. Eight of the exonic mutations lead to amino acid exchanges, causing hypomorph or null alleles. One new variation affects a splice site, which is also predicted to result in a null allele. All potential pathological alterations were present on one allele only. In six out of the 11 patients the phenotype of CSID could be explained by compound heterozygosity.     

Mechanism of ret dysfunction by Hirschsprung mutations affecting its extracellular domain

Hirschsprung disease (HSCR) is a congenital disorder associated with the absence of intrinsic ganglion cells in the distal gastrointestinal tract. Recently, many missense, nonsense and frameshift mutations of the ret proto-oncogene were found in familial and sporadic cases of HSCR. Consistent with the view that the HSCR phenotype is the result of inactivation of Ret, the missense mutations detected in the tyrosine kinase domain were demonstrated to result in a marked decrease of the kinase activity of Ret. However, the effects of missense mutations found in the extracellular domain remain unknown. We now report that five mutations in the extracellular domain examined inhibit transport of the Ret protein to the plasma membrane. As a consequence, they significantly decreased the transforming activity of Ret with multiple endocrine neoplasia (MEN) 2A mutation for which cell surface expression is required. Our results also demonstrated that long segment HSCR mutations more severely impair transport of Ret to the plasma membrane than a short segment HSCR mutation, suggesting that the level of its cell surface expression may correlate to the HSCR phenotype.      

Understanding N‐Acetyl‐L‐Glutamate Synthase Deficiency: Mutational Spectrum,Impact of Clinical Mutations on Enzyme Functionality,and Structural Considerations

N‐acetyl‐L‐glutamate synthase (NAGS) deficiency (NAGSD), the rarest urea cycle defect, is clinically indistinguishable from carbamoyl phosphate synthetase 1 deficiency, rendering the identification of NAGS gene mutations key for differentiation, which is crucial, as only NAGSD has substitutive therapy. Over the last 13 years, we have identified 43 patients from 33 families with NAGS mutations, of which 14 were novel. Overall, 36 NAGS mutations have been found so far in 56 patients from 42 families, of which 76% are homozygous for the mutant allele. 61% of mutations are missense changes. Lack or decrease of NAGS protein is predicted for ～1/3 of mutations. Missense mutations frequency is inhomogeneous along NAGS: null for exon 1, but six in exon 6, which reflects the paramount substrate binding/catalytic role of the C‐terminal domain (GNAT domain). Correspondingly, phenotypes associated with missense mutations mapping in the GNAT domain are more severe than phenotypes of amino acid kinase domain‐mapping missense mutations. Enzyme activity and stability assays with 12 mutations introduced into pure recombinant Pseudomonas aeruginosa NAGS, together with in silico structural analysis, support the pathogenic role of most NAGSD‐associated mutations found. The disease‐causing mechanisms appear to be, from higher to lower frequency, decreased solubility/stability, aberrant kinetics/catalysis, and altered arginine modulation.     

Novel mutations and genotype-phenotype relationships in 107 families with Fukuyama-type congenital muscular dystrophy (FCMD).

Fukuyama-type congenital muscular dystrophy (FCMD), one of the most common autosomal recessive disorders in the Japanese population, is characterized by congenital muscular dystrophy in combination with cortical dysgenesis (micropolygyria). Recently, we identified, on chromosome 9q31, the gene responsible for FCMD, which encodes a novel 461 amino acid protein which we have termed fukutin. Most FCMD-bearing chromosomes examined to date (87%) have been derived from a single ancestral founder, whose mutation consisted of a 3 kb retrotransposal insertion in the 3' non-coding region of the fukutin gene. FCMD is the first human disease known to be caused primarily by an ancient retrotransposal integration. We under-took a systematic analysis of the FCMD gene in 107 unrelated patients, and identified four novel non-founder mutations in five of them: one missense, one nonsense, one L1 insertion and a 1 bp insertion. The frequency of severe phenotypes, including Walker-Walberg syndrome-like manifestations such as hydrocephalus and microphthalmia, was significantly higher among probands who were compound heterozygotes carrying a point mutation on one allele and the founder mutation on the other, than it was among probands who were homozygous for the 3 kb retrotransposon. Remarkably, we detected no FCMD patients with non-founder (point) mutations on both alleles of the gene, and suggest that such cases might be embryonic-lethal. This could explain why few FCMD cases are reported in non-Japanese populations. Our results provided strong evidence that loss of function of fukutin is the major cause of FCMD, and appeared to shed some light on the mechanism responsible for the broad clinical spectrum seen in this disease.