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PubMed2001

Defective intracellular transport and processing of JAG1 missense mutations in Alagille syndrome.

Jagged1 (JAG1) is a cell surface ligand in the Notch signaling pathway and mutations in this gene cause Alagille syndrome (AGS). JAG1 mutations have been identified in 60-70% of AGS patients studied, and these include total gene deletions ( approximately 6%), protein-truncating mutations (insertions, deletions and nonsense mutations) (82%) and missense mutations (12%). Based on the finding that total JAG1 deletions cause AGS, haploinsufficiency has been hypothesized to be a mechanism for disease causation; however, the mechanism by which missense mutations cause disease is not understood. To date, 25 unique missense mutations have been observed in AGS patients. Missense mutations are non-randomly distributed across the protein with clusters at the 5' end of the protein, in the conserved DSL domain, and two clusters within the EGF repeats. To understand the effect of the missense mutations on protein localization and function, we have studied four missense mutations (R184H, L37S, P163L and P871R). In two assays of JAG1 function, R184H and L37S are associated with loss of Notch signaling activity relative to wild-type JAG1. Neither R184H or L37S is present on the cell surface and both are abnormally glycosylated. Furthermore, these mutations lead to abnormal accumulation of the protein, possibly in the endoplasmic reticulum. Both P163L and P871R are associated with normal levels of Notch signaling activity and are present on the cell surface, consistent with these changes being polymorphisms rather than disease-causing mutations.

3T3 Cells↗
PubMed1999

Subcortical band heterotopia in rare affected males can be caused by missense mutations in DCX (XLIS) or LIS1.

Subcortical band heterotopia (SBH) are bilateral and symmetric ribbons of gray matter found in the central white matter between the cortex and the ventricular surface, which comprises the less severe end of the lissencephaly (agyria-pachygyria-band) spectrum of malformations. Mutations in DCX (also known as XLIS ) have previously been described in females with SBH. We have now identified mutations in either the DCX or LIS1 gene in three of 11 boys studied, demonstrating for the first time that mutations of either DCX or LIS1 can cause SBH or mixed pachygyria-SBH (PCH-SBH) in males. All three changes detected are missense mutations, predicted to be of germline origin. They include a missense mutation in exon 4 of DCX in a boy with PCH-SBH (R78H), a different missense mutation in exon 4 of DCX in a boy with mild SBH and in his mildly affected mother (R89G) and a missense mutation in exon 6 of LIS1 in a boy with SBH (S169P). The missense mutations probably account for the less severe brain malformations, although other patients with missense mutations in the same exons have had diffuse lissencephaly. Therefore, it appears likely that the effect of the specific amino acid change on the protein determines the severity of the phenotype, with some mutations enabling residual protein function and allowing normal migration in a larger proportion of neurons. However, we expect that somatic mosaic mutations of both LIS1 and DCX will also prove to be an important mechanism in causing SBH in males.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed2004

Disease-causing missense mutations in NPHS2 gene alter normal nephrin trafficking to the plasma membrane.

BACKGROUND: Podocin is a membrane-integrated protein that is located at the glomerular slit diaphragm and directly interacts with nephrin. The gene encoding podocin, NPHS2, is mutated in patients with autosomal-recessive steroid-resistant nephrotic syndrome (SRN). In order to study a potential pathomechanism of massive proteinuria in patients with SRN, we have investigated the trafficking and subcellular localization of five common disease-causing missense mutants of human podocin. METHODS: Site-directed mutagenesis was applied to generate cDNA constructs encoding five different missense mutations of human podocin (P20L, G92C, R138Q, V180M, and R291W). To identify the subcellular localization of each mutant in transfected human embryonic kidney (HEK)293 cells, we have generated and characterized a rabbit polyclonal antibody against the human podocin. Specificity of the antibody was determined by light and immunoelectron microscopy, as well as immunoblot analysis using human glomeruli. Confocal microscopy was applied to determine subcellular localization of the wild-type and the mutated podocin molecules, as well as wild-type nephrin in transfected cells. Immunoprecipitation and pull-down studies were carried out to investigate the molecular interaction of podocin mutants and wild-type nephrin. RESULTS: Immunofluorescence and confocal microscopy showed that wild-type podocin located to the plasma membrane when expressed in HEK293 cells. Two missense mutations, P20L and G92C, located at the N-terminus part of the molecule, were also present at the plasma membrane, indicating that these mutations did not affect the subcellular localization of the mutated podocin molecules. In contrast, subcellular localization of three other missense mutants located in the proximal C-terminus part of the protein was drastically altered, in which R138Q was retained in the endoplasmic reticulum (ER), V180M formed inclusion bodies in the cytoplasm, and the R291W mutant was trapped both in the ER and in small intracellular vesicles. Interestingly, this abnormal subcellular localization of podocin missense mutants also resulted in alteration in protein trafficking of wild-type nephrin in cotransfected cells through the strong protein binding between both molecules. CONCLUSION: In patients with SRN, some missense mutations in the NPHS2 gene not only lead to misfolding and mislocalization of the mutated podocin, but they can also interfere with slit diaphragm structure and function by altering the proper trafficking of nephrin to the plasma membrane.

Cell Line↗
PubMed2005

Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral.

BACKGROUND: Genetic testing for hereditary cancer syndromes contributes to the medical management of patients who may be at increased risk of one or more cancers. BRCA1 and BRCA2 testing for hereditary breast and ovarian cancer is one such widely used test. However, clinical testing methods with high sensitivity for deleterious mutations in these genes also detect many unclassified variants, primarily missense substitutions. METHODS: We developed an extension of the Grantham difference, called A-GVGD, to score missense substitutions against the range of variation present at their position in a multiple sequence alignment. Combining two methods, co-occurrence of unclassified variants with clearly deleterious mutations and A-GVGD, we analysed most of the missense substitutions observed in BRCA1. RESULTS: A-GVGD was able to resolve known neutral and deleterious missense substitutions into distinct sets. Additionally, eight previously unclassified BRCA1 missense substitutions observed in trans with one or more deleterious mutations, and within the cross-species range of variation observed at their position in the protein, are now classified as neutral. DISCUSSION: The methods combined here can classify as neutral about 50% of missense substitutions that have been observed with two or more clearly deleterious mutations. Furthermore, odds ratios estimated for sets of substitutions grouped by A-GVGD scores are consistent with the hypothesis that most unclassified substitutions that are within the cross-species range of variation at their position in BRCA1 are also neutral. For most of these, clinical reclassification will require integrated application of other methods such as pooled family histories, segregation analysis, or validated functional assay.

Amino Acid Sequence↗
PubMed2026

Systematic functional evaluation of CNGA1 missense variants associated with retinitis pigmentosa.

BACKGROUND: Missense variants are frequently classified as variants of uncertain significance (VUS) according to the guidelines of the American College of Medical Genetics and Genomics and the Association of Molecular Pathology (ACMG/AMP). Consequently, disease relevance remains elusive, impeding molecular genetic diagnostics, patients` and family genetic counseling, and identification of patients eligible for clinical trials. Functional studies are critical for resolving the clinical significance of VUS. CNGA1 encodes the main subunit of the rod cyclic nucleotide-gated (CNG) channel, a vital component of the phototransduction cascade. Variants in CNGA1 are a rare cause of autosomal recessive retinitis pigmentosa and a phase I/II gene augmentation trial (NCT06291935) is currently ongoing highlighting the necessity to differentiate benign from pathogenic variants. METHODS: CNGA1 missense variants compiled from retinal disease patient cohorts, public databases and literature were functionally investigated using a medium-throughput aequorin-based assay and in vitro minigene splice assays for predicted exonic spliceogenic variants. Functional data were correlated with the in silico prediction of five variant effect predictors (VEPs) and applied to support or revise variants' ACMG/AMP classification. RESULTS: Data mining revealed 86 missense CNGA1 variants - including three novel - most of them lacking functional data; 65.1% of the variants were initially classified as VUS. The aequorin-based assay showed that 72.1% of tested variants significantly impaired CNG channel function and were classified as functionally abnormal, while 23.3% were functionally normal and 5% remained functionally uncertain. Correlation of the functional data with in silico predictions identified AlphaMissense and CPT-1 to be the most suitable tools for assessing CNGA1 missense variants. Using in vitro minigene splice assays, two putative missense variants were shown to induce missplicing. Based on the functional findings, 62.1% of the variants initially classified as VUS were re-categorized as likely pathogenic or likely benign. Furthermore, 93.3% of the variants initially classified as likely pathogenic showed an effect on CNGA1 channel function, confirming their disease relevance and supporting their reclassification as pathogenic. CONCLUSION: This study represents the first comprehensive functional assessment of disease-associated CNGA1 missense variants, thus significantly advancing the understanding of their disease relevance and improving molecular genetic diagnostics in patients.

Humans↗
PubMed1992

Missense mutations and the magnitude of functional deficit: the example of factor IX.

Some missense changes are compatible with normal protein function while others compromise essential aspects of protein maturation, specific activity, or stability. For those missense changes that alter function in the intact organism, how likely is it for the mutated protein to retain appreciable residual activity? By genetic analysis of patients with hemophilia B of known severity, this question can be addressed for missense mutations that reduce factor IX activity by fourfold or more below the average. We estimate that missense changes cause only 59% of moderate and severe disease, but these mutations are almost always (95%) of independent origin. In contrast, missense mutations are found in virtually all (97%) families with mild disease, but only a minority of these (41%) are of independent origin. From the aggregate data, we estimate that most (71%) of the independent deleterious missense mutations cause at least a 20-fold decrease in factor IX activity.

Factor IX↗
PubMed1993

Concentrating missense mutations in core gene of hepatitis B virus. Evidence for adaptive mutation in chronic hepatitis B virus infection.

To elucidate the temporal relationship between liver damage and mutation(s) in hepatitis B virus core gene, serial sera from a progressive liver disease patient and an asymptomatic carrier were studied. By direct sequencing, missense mutations in the core gene were only found in serum from the progressive liver disease patient during the period with frequent exacerbation. Using methods of cloning and sequencing, missense mutations were also found in clones derived from the progressive liver disease patient at a relatively early phase, but strains with a missense mutation from earlier sera did not exist in sera of a later period. Furthermore, there was a tendency of concentrating missense mutations in clones derived from the progressive liver disease patient. These data suggested that missense mutations in the core gene that occurred at an earlier phase might evoke an immune response to eliminate mutated virus and that concentrating missense mutations during a phase of exacerbation might be a result of adaptive mutation.

Adaptation, Physiological↗
PubMed1976

Suppressor-induced structural changes in a missense L-ribulokinase of Escherichia coli.

A suppressor mutation specific for a missense codon in the L-ribulokinase structural gene of the L-arabinose operon of Escherichia coli B/r enhanced L-arabinose utilization by the strain containing the missense codon. Electrophoretic comparisons of the wild-type, missense, and suppressed missense L-ribulokinases indicated that the suppressor changed the structure of the missense kinase, thereby increasing its catalytic activity. Hyperinducibility imposed on an operator-distal gene by the missense codon was not affected by the suppressor mutation.

Arabinose↗
PubMed2026

A novel PKHD1 missense variant disrupting splicing in a fetus with Caroli disease.

BACKGROUND: Caroli disease (CD) is a rare inherited disorder characterized by dilatation of intrahepatic bile ducts, and prenatal diagnosis of this disease is extremely rare. PKHD1 is the only known causative gene, yet the pathogenicity of most missense variants remains unclear. METHODS: Exome sequencing (ES) was performed on a fetus with clinical features of CD. Candidate variants were validated by Sanger sequencing in the family. The impact of the novel missense variant on pre-mRNA splicing was assessed using minigene assays, and structural modeling of the PKHD1 protein was conducted with AlphaFold 3. RESULTS: At 23 weeks of gestation, the fetus showed hepatic cysts on ultrasound and a "central dot" sign on MRI, suggesting a diagnosis of CD. The fetus also exhibited features of autosomal recessive polycystic kidney disease and oligohydramnios. ES identified and Sanger sequencing confirmed three PKHD1 variants: a paternal nonsense variant c.5323C>T; p.(Arg1775*), and two maternal missense variants c.6682G>C; p.(Glu2228Gln) and c.8012G>T; p.(Arg2671Leu). The variant c.6682G>C is novel and minigene assays demonstrated that it caused exon 40 skipping, leading to an in‑frame deletion (c.6491_6682del; p.(Gly2164_Arg2227del)). Structural modeling predicts that this deletion lies within a large β‑barrel domain and may compromise its structural stability. Conclusion We characterize a novel missense variant that causes aberrant splicing of PKHD1 in CD. This finding underscores the necessity of functional analysis for evaluating the pathogenicity of missense variants, especially those at the last nucleotide of an exon. Our study expands the mutation spectrum of PKHD1 and provides insights into genotype‑phenotype correlations.

Humans↗
PubMed2005

Analysis of missense variants in the PKHD1-gene in patients with autosomal recessive polycystic kidney disease (ARPKD).

Autosomal recessive polycystic kidney disease (ARPKD) is a severe form of polycystic kidney disease characterized by enlarged kidneys and congenital hepatic fibrosis. Given the poor prognosis for the majority of children with the severe perinatal ARPKD phenotype, there is a regular request for prenatal testing. ARPKD is caused by mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, which consists of 86 exons that are variably assembled into a number of alternatively spliced transcripts. The longest transcript, comprising 67 exons, encodes the protein fibrocystin/polyductin. We have set up mutation analysis by direct sequencing of these 67 exons. In 39 mainly Dutch families we identified: 11 nonsense mutations, 15 deletions/insertions, 5 splice site mutations, and 39 missense mutations. To classify missense variants we combined evolutionary conservation, using the human, chimpanzee, dog, mouse, chicken and frog Pkhd1 sequences, with the Grantham score for chemical differences. Thirty-three missense mutations were considered pathogenic and seven were classified as rare, probably pathogenic variants. In addition to sequence analysis, multiplex ligation-dependent probe amplification (MLPA) was used to identify multiple exon deletions. However, no large deletions in the PKHD1 gene were identified. In 31 index patients two mutations were found, in 6 patients one mutation was found, leading to a mutation detection rate of 87%. The analysis of amino acid conservation as well as applying the Grantham score for chemical differences allowed us to determine the pathogeneity for nearly all new missense mutations and thus proved to be useful tools to classify missense variants.

Amino Acid Sequence↗
PubMed2004

Lattice corneal dystrophy associated with the Ala546Asp and Pro551Gln missense changes in the TGFBI gene.

PURPOSE: To report a phenotypic variant of lattice corneal dystrophy associated with two missense changes, Ala546Asp and Pro551Gln, in the transforming growth factor-beta-induced gene (TGFBI). DESIGN: Experimental study. METHODS: Genomic DNA was obtained from the proband as well as affected and unaffected family members. Exons 4, 11, 12, and 14 of the TGFBI gene were amplified and sequenced. Additionally, a corneal button excised from the proband was examined by light and transmission electron microscopy. Haplotype analysis was performed on the proband's family and members of a previously identified pedigree with the same TGFBI gene missense changes. RESULTS: Bilateral, symmetric, radially arranged, branching refractile lines within and surrounding an area of central anterior stromal haze were noted in the proband. Multiple polymorphic, refractile deposits were noted in the mid and posterior stroma in both the proband and her daughter. Light and electron microscopic analyses demonstrated amyloid and excluded the presence of deposits characteristic of granular corneal dystrophy. Screening of TGFBI exon 12 in the proband and her affected daughter revealed two missense changes, Ala546Asp and Pro551Gln (both absent in 250 control chromosomes). Haplotype analysis suggested that the mutations in this family and in a previously identified pedigree reflect a founder effect, rather than an independent occurrence. CONCLUSIONS: We present a phenotypic variant of lattice corneal dystrophy associated with the Ala546Asp and Pro551Gln missense changes in exon 12 of the TGFBI gene. A common ancestor appears to account for the missense mutations observed in this pedigree and in a previously reported family.

Adult↗
PubMed2004

MSH6 missense mutations are often associated with no or low cancer susceptibility.

Mismatch repair (MMR) deficiency in tumours from patients with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is mainly caused by mutations in the MLH1, MSH2, and MSH6 genes. A major challenge in the clinical management of patients with suspected HNPCC is the frequent occurrence of missense mutations in MSH6. These can be considered neither deleterious nor clinically innocent a priori. To assess their significance we studied five novel MSH6 missense mutations in six patients derived from a series of consecutive endometrial and colorectal cancer patients selected for study after their tumours were determined to be microsatellite unstable. We tested each mutated protein for heterodimerisation with MSH2 and for in vitro MMR capability. Four mutations (R128L, P623L, K728T, G881K+S) showed no impairment of these functions while the fifth (E1193K) displayed marked impairment of both functions. These results, taken together with our previous similar findings concerning six other missense mutations in MSH6, allow us to conclude that many or most missense changes in MSH6 likely are clinically innocent, whereas some missense changes such as E1193K, which lead to impaired MMR, are likely to be clinically significant, but have low penetrance.

Base Pair Mismatch↗
PubMed2006

Pathogenicity of MSH2 missense mutations is typically associated with impaired repair capability of the mutated protein.

BACKGROUND & AIMS: Inherited deleterious mutations in mismatch repair genes MLH1, MSH2, and MSH6 predispose to hereditary nonpolyposis colorectal cancer. A major diagnostic challenge is the difficulty in evaluating the pathogenicity of missense mutations. Previously we showed that most missense variants in MSH6 do not impair MMR capability and are associated with no or low cancer susceptibility, whereas in MLH1, functional studies distinguished nontruncating mutations with severe defects from those not or slightly impaired in protein expression or function. The present study was undertaken to evaluate the pathogenicity of inherited missense mutations in MSH2. METHODS: Fifteen mutated MSH2 proteins including 14 amino acid substitutions and one in-frame deletion were tested for expression/stability, MSH2/MSH6 interaction, and repair efficiency. The genetic and biochemical data were correlated with the clinical data. Comparative sequence analysis was performed to assess the value of sequence homology as a tool for predicting functional results. RESULTS: None of the studied MSH2 mutations destroyed the protein or abolished MSH2/MSH6 interaction, whereas 12 mutations impaired the repair capability of the protein. Comparative sequence analysis correctly predicted functional studies for 13 of 14 amino acid substitutions. CONCLUSIONS: Interpretation was pathogenic for 12, nonpathogenic for 2, and contradictory for 1 mutation. The pathogenicity could not be distinguished unambiguously by phenotypic characteristics, although correlation between the absence of staining for MSH2 and pathogenicity of the missense mutation was notable. Unlike in MSH6 and MLH1, the pathogenicity of missense mutations in MSH2 was always associated with impaired repair capability of the mutated protein.

Adaptor Proteins, Signal Transducing↗
PubMed1999

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.

Amino Acid Sequence↗
PubMed2000

Biochemical and structural analysis of missense mutations in N-acetylgalactosamine-6-sulfate sulfatase causing mucopolysaccharidosis IVA phenotypes.

Mucopolysaccharidosis IVA (MPS IVA; OMIM#253000), a lysosomal storage disorder caused by a deficiency of N -acetylgalactosamine-6-sulfate sulfatase (GALNS), has variable clinical phenotypes. To date we have identified 65 missense mutations in the GALNS gene from MPS IVA patients, but the correlation between genotype and phenotype has remained unclear. We studied 17 missense mutations using biochemical approaches and 32 missense mutations, using structural analyses. Fifteen missense mutations and two newly engineered active site mutations (C79S, C79T) were characterized by transient expression analysis. Mutant proteins, except for C79S and C79T, were destabilized and detected as insoluble precursor forms while the C79S and C79T mutants were of a soluble mature size. Mutants found in the severe phenotype had no activity. Mutants found in the mild phenotype had a considerable residual activity (1.3-13.3% of wild-type GALNS activity). Sulfatases, including GALNS, are members of a highly conserved gene family sharing an extensive sequence homology. Thus, a tertiary structural model of human GALNS was constructed from the X-ray crystal structure of N -acetylgalacto-samine-4-sulfatase and arylsulfatase A, using homology modeling, and 32 missense mutations were investigated. Consequently, we propose that there are at least three different reasons for the severe phenotype: (i) destruction of the hydrophobic core or modification of the packing; (ii) removal of a salt bridge to destabilize the entire conformation; (iii) modification of the active site. In contrast, mild mutations were mostly located on the surface of the GALNS protein. These studies shed further light on the genotype-phenotype correlation of MPS IVA and structure-function relationship in the sulfatase family.

Amino Acid Sequence↗
PubMed2004

Defective trafficking of nephrin missense mutants rescued by a chemical chaperone.

The nephrin gene (NPHS1) is mutated in congenital nephrotic syndrome of the Finnish type. Most mutations found in non-Finnish patients are missense mutations. The most common consequence of missense mutations in congenital nephrotic syndrome is a defect in intracellular transport and retention of the mutant proteins in the endoplasmic reticulum (ER), possibly as a result of misfolding and unfavored conformation. Because sodium 4-phenylbutyrate has been shown to function as a chemical chaperone and to correct the cellular trafficking of several mislocalized or misfolded mutant plasma membrane proteins, the effects of this compound on the missense mutants identified in patients with congenital nephrotic syndrome of the Finnish type were investigated. This study was performed using human embryonic kidney 293 cells stably expressing wild-type or missense nephrin mutants trapped in the ER. Immunofluorescence microscopy and cell surface biotinylation showed that treatment with sodium 4-phenylbutyrate rescued several of the missense mutants from the ER to the cell surface. All of the rescued mutants were found to be able to interact with Neph1. Furthermore, their tyrosine phosphorylation was rapidly induced by clustering with anti-nephrin antibodies, suggesting that the rescued mutants may be functionally intact.

Biological Transport↗
PubMed2026

A Second Report of a Missense Variant in AMMECR1 Causing Midface Hypoplasia, Hearing Impairment, Elliptocytosis, and Nephrocalcinosis: Case Report and Literature Review.

Pathogenic variants in AMMECR1 have been associated with a rare multisystem disorder characterized by midface hypoplasia, hearing impairment, elliptocytosis, and nephrocalcinosis (MFHIEN). To date, most reported cases involve copy number variants or presumed loss-of-function alterations, with only a single prior report describing a missense variant supported by functional studies. Here, we report a patient with a heterozygous de novo AMMECR1 missense variant, NM_015365.3:c.649G>A p.(Val217Met) presenting with clinical features consistent with MFHIEN, including midface hypoplasia, partial hearing impairment, nephrocalcinosis, and elliptocytosis identified on peripheral blood smear. Comparative review of the literature highlights that while previously reported missense variants in AMMECR1 demonstrated altered intranuclear protein distribution and reduced expression in functional assays, clinical evidence supporting pathogenicity of non-truncating variants remains limited. The phenotypic overlap between our patient and prior report strengthens the association between missense variations and the MFHIEN phenotype. Our findings support the pathogenic relevance of missense variation in AMMECR1 and emphasize the importance of integrating detailed phenotyping, including hematologic evaluation, with genomic data in the diagnosis of rare multisystem disorders. Additional cases and functional studies are needed to clarify genotype-phenotype correlations and underlying disease mechanisms.

Humans↗
PubMed2002

Pathogenicity of missense and splice site mutations in hMSH2 and hMLH1 mismatch repair genes: implications for genetic testing.

BACKGROUND: In hereditary non-polyposis colorectal cancer, over 90% of the identified mutations are in two genes, hMSH2 and hMLH1. A large proportion of the mutations detected in these genes are of the missense type which may be either deleterious mutations or harmless polymorphisms. AIM: To investigate whether nine missense and one splice site mutation of hMLH1 and hMSH2, in 10 kindreds with a familial history of colorectal cancer or young age of onset, could be interpreted as pathogenic. METHODS: Clinical and genetic characteristics were collected: (i) evolutionary conservation of the codon involved; (ii) type of amino acid change; (iii) occurrence of mutation in healthy controls; (iv) cosegregation of mutation with disease phenotype; (v) functional consequences of gene variant; and (vi) microssatellite instability and immunoexpression of hMSH2 and hMLH1 analysis. RESULTS: Seven different missense and one splice site mutation were identified. Only 1/8 was found in the control group, 2/7 occurred in conserved residues, and 5/7 resulted in non-conservative changes. Functional studies were available for only 2/8 mutations. Segregation of the missense variant with disease phenotype was observed in three kindreds. CONCLUSION: In the majority of families included, there was no definitive evidence that the missense or splice site alterations were causally associated with an increased risk of developing colorectal cancer. Until further evidence is available, these mutational events should be regarded and interpreted carefully and genetic diagnosis should not be offered to these kindreds.

Adaptor Proteins, Signal Transducing↗