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PubMed2001

Late-onset Stargardt disease is associated with missense mutations that map outside known functional regions of ABCR (ABCA4).

Based on recent studies of the photoreceptor-specific ABC transporter gene ABCR (ABCA4) in Stargardt disease (STGD1) and other retinal dystrophies, we and others have developed a model in which the severity of retinal disease correlates inversely with residual ABCR activity. This model predicts that patients with late-onset STGDI may retain partial ABCR activity attributable to mild missense alleles. To test this hypothesis, we used late-onset STGDI patients (onset: > or =35 years) to provide an in vivo functional analysis of various combinations of mutant alleles. We sequenced directly the entire coding region of ABCR and detected mutations in 33/50 (66%) disease chromosomes, but surprisingly, 11/33 (33%) were truncating alleles. Importantly, all 22 missense mutations were located outside the known functional domains of ABCR (ATP-binding or transmembrane), whereas in our general cohort of STGDI subjects, alterations occurred with equal frequency across the entire protein. We suggest that these missense mutations in regions of unknown function are milder alleles and more susceptible to modifier effects. Thus, we have corroborated a prediction from the model of ABCR pathogenicity that (1) one mutant ABCR allele is always missense in late-onset STGD1 patients, and (2) the age-of-onset is correlated with the amount of ABCR activity of this allele. In addition, we report three new pseudodominant families that now comprise eight of 178 outbred STGD1 families and suggest a carrier frequency of STGD1-associated ABCR mutations of about 4.5% (approximately 1/22).

ATP-Binding Cassette Transporters↗
PubMed2005

A novel missense mutation in a Japanese patient with gelatinous droplike corneal dystrophy.

PURPOSE: To report a novel missense mutation in TACSTD2 gene, L186P, responsible for gelatinous droplike dystrophy (GDLD). DESIGN: Case report and experimental study. METHOD: A 10-year-old Japanese boy suffering from typical GDLD was studied. A 1.1-kb DNA fragment of the TACSTD2 gene was amplified and analyzed using a molecular biological method. cDNA from the patient's cornea was also analyzed to determine which allele was expressed in the patient's corneal epithelium. RESULTS: Sequence analysis revealed that the patient is a compound heterozygote for the Q118X mutation and the L186P, the first missense mutation found in Japanese GDLD. Polymerase chain reaction-restriction fragment length polymorphism analysis from cDNA of patient's cornea revealed that the L186P missense mutation allele is expressed in the patient's corneal epithelium. CONCLUSION: We describe a novel mutation in one case of Japanese GDLD. The results confirm that the missense mutation L186P in the TACSTD2 gene is also responsible for the GDLD phenotype.

Antigens, Neoplasm↗
PubMed2005

Early onset, non fluctuating spinocerebellar ataxia and a novel missense mutation in CACNA1A gene.

Mutations in the brain-specific P/Q type Ca2+ channel alpha1 subunit gene, CACNA1A, have been identified in three clinically distinct disorders, spinocerebellar ataxia type 6 (SCA6), episodic ataxia type 2 (EA2), and familial hemiplegic migraine type 1 (FHM1). SCA6 is associated with small expansions of a CAG repeat at the 3' end of the gene, while point mutations are mostly responsible for its two allelic disorders, FHMI and EA2. From the electrophysiological point of view, while FHMI mutations lead to a gain of function [Tottene A, Fellin T, Pagnutti S, Luvisetto S, Striessnig J, Fletcher C, et al. Familial hemiplegic migraine mutations increase Ca2+ influx through single human CaV2.1 channels and decrease maximal CaV2.1 current density in neurons. Proc Natl Acad Sci 99 (20) (2002) 13284-13289.], EA2 mutations usually generate a loss of channel function [Guida S, Trettel F, Pagnutti S, Mantuano E, Tottene A, Veneziano L, et al. Complete loss of P/Q calcium channel activity caused by a CACNA1A missense mutation carried by patients with episodic ataxia type 2. Am J Hum Genet 68 (3) (2001) 759-764, Wappl E, Koschak A, Poteser M, Sinnegger MJ, Walter D, Eberhart A, et al. Functional consequences of P/Q-type Ca2+ channel Cav2.1 missense mutations associated with episodic ataxia type 2 and progressive ataxia. J Biol Chem 277 (9) (2002) 6960-6966.]. In the present study, we describe a child affected by permanent non-fluctuating limb and trunk ataxia with a quite early age of onset. Interestingly, the size of the CACNA1A triplet repeat region in the patient is within the normal range while he carries a novel de novo missense mutation in this gene, p.R1664Q. Although functional data are not available, based on the literature data indicating that severe reductions in P/Q-type channel activity favour episodic and/or progressive ataxic symptoms [Wappl E, Koschak A, Poteser M, Sinnegger MJ, Walter D, Eberhart A, et al. Functional consequences of P/Q-type Ca2+ channel Cav2.1 missense mutations associated with episodic ataxia type 2 and progressive ataxia. J Biol Chem 2002;277(9):6960-6966.], we hypothesize that the functional consequence of the mutation here identified is a partial loss of the Ca channel function. In conclusion, the clinical and molecular findings reported here suggest the opportunity to screen for point mutation in this gene, even patients with a clinical phenotype for some aspects slightly different from the typical picture more commonly associated to SCA6, EA2 or FHM1 diseases.

Age of Onset↗
PubMed2004

Identification of paraoxonase 3 gene (PON3) missense mutations in a population of southern Italy.

PON gene family includes at least three members termed PON1, PON2 and PON3, and it is mapped on human chromosome 7q21-q22. PON1 and PON3 gene products are constituents of high density lipoprotein (HDL) and have many enzymatic properties and antioxidant activity. PONs are proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. PON1 and PON2 genes have missense polymorphisms, but, to date, no missense variants are reported in PON3 gene. In this work we explored the existence of genetic variants within the PON3 coding sequences. Five point mutations were identified by direct sequencing of genomic DNA derived from 250 randomly selected DNA samples of 1143 blood donors living in southern Italy. Three were silent mutations, while two were missense mutations that give rise to amino acid substitutions at positions 311 (S>T) and 324 (G>D). The missense variations in the DNA of the 1143 samples had frequencies of 0.22% (5 out of 2286 alleles) for the S311T mutation, and 0.57% (13 out of 2286 alleles) for the G324D mutation. The effect of these variants on the metabolic activity of paraoxonase 3 remains to be further evaluated.

Alleles↗
PubMed2002

Elevated frequency of ATM gene missense mutations in breast cancer relative to ethnically matched controls.

Studies of families of patients with ataxia telangiectasia (A-T) show an increased risk of breast cancer in heterozygous A-T carriers. However, expected increased levels of mutations in the ATM gene among unselected breast cancer patients have not been found to date. Previous methods of mutation detection were biased toward the detection of truncating mutations, and single nucleotide substitutions were likely to have been underreported. In this study, genomic DNA from 43 breast cancer patients and 43 control individuals were scanned for mutations in the entire ATM coding region (exons 4-65) and adjacent intronic splice regions (three megabases total) using detection of virtually all mutation-single-strand conformation polymorphism (SSCP), a modification of SSCP with sufficient redundancy to detect virtually all mutations. Excluding a polymorphism found commonly in cases and controls, there were missense changes in 12 breast cancer patients, one of whom also had a protein truncating mutation, versus six controls (P=0.09). When all structural changes common to the cases and controls were excluded, missense or truncating changes were found in 10 cases compared to two in controls (P=0.013). The background of missense changes in controls is high. There is a trend towards elevation of all structural changes in cases, but the results are not statistically significant. Cohort-specific structural changes are significantly more prevalent in the breast cancer patients. The data are compatible with certain missense mutations in ATM predisposing to breast cancer.

Adult↗
PubMedDate not supplied

Assessment of pathogenicity criteria for constitutional missense mutations of the hereditary nonpolyposis colorectal cancer genes MLH1 and MSH2.

To determine the role played by MLH1 and MSH2 missense variants in cancer susceptibility, we have investigated the following genetic and biological characteristics associated with six MLH1 and four MSH2 missense changes identified in Italian hereditary nonpolyposis colorectal cancer (HNPCC) families: co-segregation with disease phenotype and/or bonafide pathogenetic mutations; presence of the variant in healthy control subjects; evolutionary conservation of the involved aminoacid and type of aminoacid change; and presence/absence of microsatellite instability (MSI) in tumour DNA. Overall, nine variants did not fulfil > or = 2 pathogenicity criteria. MSI was investigated in tumour samples from carriers of nine different missense mutations. Only 3/9 variants were associated with MSI in tumour DNA. In addition, four variants were not present in affected pedigree members, and five variants were observed in the control population. Based upon these results, we conclude that most MLH1 and MSH2 missense changes are unlikely to act as major causative factors in colorectal cancer susceptibility and development.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Assessing the pathogenicity of MLH1 missense mutations in patients with suspected hereditary nonpolyposis colorectal cancer: correlation with clinical, genetic and functional features.

Assessing the pathogenicity of missense mutations of MLH1 and MSH2 is critical to counsel patients with suspected hereditary nonpolyposis colorectal cancer (HNPCC). Approximately 32% of all MLH1 mutations and 18% of MSH2 mutations are missense variants which often have an uncertain genetic significance. To assess the pathogenicity of four MLH1 missense mutations which were found in five patients with suspected HNPCC, P648S (CCC --> TCC), L559R (CTG --> CGG), K618A (AAG --> GCG), Y646C (TAT --> TGT), we studied their ability to disrupt MLH1 protein function and their relationship with all those clinical, genetic and pathological features which are typical of this syndrome. Our results indicated that the P648S and L559R mutations were probably pathogenic because they disrupted MLH1 protein interaction with its partner PMS2 in vitro and abolished MLH1 expression in HCT116 cells. In addition these variants were associated with features often found in HNPCC patients: in particular high microsatellite instability, occurrence of high grade tumours and, in one case, strong family history. The pathogenicity of the K618A and Y646C mutations was questionable as their correlation with features typical of HNPCC was low and the outcome of the functional analysis was ambiguous. These observations suggested that a clinically usable assessment of the pathogenicity of MLH missense variants can be achieved through the analysis of multiple mutation characteristics among which loss of protein function, occurrence of microsatellite instability and family history seemed to have a predominant role.

Adaptor Proteins, Signal Transducing↗
PubMed2002

A missense mutation in the nephrin gene impairs membrane targeting.

BACKGROUND: NPHS1, which encodes nephrin, recently has been identified as the gene in which mutations cause congenital nephrotic syndrome of the Finnish type (CNF). We previously reported novel missense mutations of NPHS1 in a Japanese patient with CNF. However, the mechanism by which these missense mutations cause the disorder remains to be clarified. METHODS: Wild-type nephrin and mutated nephrin complementary DNA were each tagged by the green fluorescence protein (GFP) gene; the expressing vectors of the fusion protein were each transfected to human embryonic kidney 293 cells. We compared intracellular localization of mutated nephrin with that of wild-type nephrin by using GFP and immunostaining examination. RESULTS: In both wild-type and mutated nephrin (Glu(447)Lys), GFP and immunostaining resulted in a colocalized microgranular pattern along the cell membrane that indicated these recombinant proteins were located at the cell surface. Conversely, in mutated nephrin (Asp(819)Val), GFP aggregation was observed in the cytoplasm, and no fluorescence was observed at the cell membrane, indicating that recombinant mutated nephrin (Asp(819)Val) could not be distributed at the cell membrane and instead was retained in cytoplasm. CONCLUSION: We confirmed that the missense mutation GAC-to-GTC transversion leading to an Asp(819)Val caused the disorder. The present study analyzes in vitro distribution of nephrin with a missense point mutation. The analysis uses a new convenient method, construction of a nephrin-GFP fusion protein.

Blotting, Western↗
PubMed2003

Understanding missense mutations in the BRCA1 gene: an evolutionary approach.

The role of missense changes in BRCA1 in breast cancer susceptibility has been difficult to establish. We used comparative evolutionary methods to identify potential functionally important amino acid sites in exon 11 and missense changes likely to disrupt gene function, aligning sequences from 57 eutherian mammals and categorizing amino acid sites by degree of conservation. We used Bayesian phylogenetic analyses to determine relationships among orthologs and identify codons evolving under positive selection. Most conserved residues occur in a region with the highest concentration of protein-interacting domains. Rapidly evolving residues are concentrated in the RAD51-interacting domain, suggesting that selection is acting most strongly on the role of BRCA1 in DNA repair. Investigation of the functional role of missense changes in breast-cancer susceptibility should focus on 38 missense changes in conserved and 3 in rapidly evolving regions of exon 11.

Algorithms↗
PubMed2002

Functional analysis of TBX5 missense mutations associated with Holt-Oram syndrome.

TBX5 is a T-box transcription factor that plays a critical role in organogenesis. Seven missense mutations in TBX5 have been identified in patients with Holt-Oram syndrome characterized by congenital heart defects and upper limb abnormalities. However, the functional significance and molecular pathogenic mechanisms of these mutations are not clear. In this study we describe functional defects in DNA binding, transcriptional activity, protein-protein interaction, and cellular localization of mutant TBX5 with these missense mutations (Q49K, I54T, G80R, G169R, R237Q, R237W, and S252I). Mutations G80R, R237Q, and R237W represent a group of mutations that dramatically reduce DNA-binding activity of TBX5, leading to reduced transcription activation by TBX5 and the loss of synergy in transcriptional activation between TBX5 and NKX2.5. The second group of mutations includes Q49K, I54T, G169R, and S252I, which have no or moderate effect on DNA-binding activity and the function of transcription activation of TBX5 but cause the complete loss of synergistic transcription activity between TBX5 and NKX2.5. All seven missense mutations greatly reduced the interaction of TBX5 with NKX2.5 in vivo and in vitro. Immunofluorescent staining showed that wild type TBX5 was localized completely into the nucleus, but mutants were localized in both nucleus and cytoplasm. These results demonstrate that all seven missense mutations studied here are functional mutations with a spectrum of defects ranging from decreases in DNA-binding activity and transcriptional activation to the dramatic reduction of interaction between TBX5 and NKX2.5, and loss of synergy in transcriptional activation between these two proteins, as well as impairment in the nuclear localization of TBX5. These defects are likely central to the pathogenesis of Holt-Oram syndrome.

Abnormalities, Multiple↗
PubMed2001

Complete loss of P/Q calcium channel activity caused by a CACNA1A missense mutation carried by patients with episodic ataxia type 2.

Familial hemiplegic migraine, episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 are allelic disorders of the CACNA1A gene (coding for the alpha(1A) subunit of P/Q calcium channels), usually associated with different types of mutations (missense, protein truncating, and expansion, respectively). However, the finding of expansion and missense mutations in patients with EA2 has blurred this genotype-phenotype correlation. We report the first functional analysis of a new missense mutation, associated with an EA2 phenotype-that is, T-->C transition of nt 4747 in exon 28, predicted to change a highly conserved phenylalanine residue to a serine at codon 1491, located in the putative transmembrane segment S6 of domain III. Patch-clamp recording in HEK 293 cells, coexpressing the mutagenized human alpha(1A-2) subunit, together with human beta(4) and alpha(2)delta subunits, showed that channel activity was completely abolished, although the mutated protein is expressed in the cell. These results indicate that a complete loss of P/Q channel function is the mechanism underlying EA2, whether due to truncating or to missense mutations.

Amino Acid Sequence↗
PubMed2001

Functional analysis of human MLH1 and MSH2 missense variants and hybrid human-yeast MLH1 proteins in Saccharomyces cerevisiae.

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant inherited disease caused by defects in the process of DNA mismatch repair (MMR), and mutations in the hMLH1 or hMSH2 genes are responsible for the majority of HNPCC. In addition to clear loss-of-function mutations conferred by nonsense or frameshift alterations in the coding sequence or by splice variants, genetic screening has revealed a large number of missense codons with less obvious functional consequences. The ability to discriminate between a loss-of-function mutation and a silent polymorphism is important for genetic testing for inherited diseases like HNPCC where the opportunity exists for early diagnosis and preventive intervention. In this study, quantitative in vivo DNA MMR assays in the yeast Saccharomyces cerevisiae were performed to determine the functional significance of amino acid replacements observed in the human population. Missense codons previously observed in human genes were introduced at the homologous residue in the yeast MLH1 or MSH2 genes. This study also demonstrated feasibility of constructing genes that encode functional hybrid human-yeast MLH1 proteins. Three classes of missense codons were found: (i) complete loss of function, i.e. mutations; (ii) variants indistinguishable from wild-type protein, i.e. silent polymorphisms; and (iii) functional variants which support MMR at reduced efficiency, i.e. efficiency polymorphisms. There was a good correlation between the functional results in yeast and available human clinical data regarding penetrance of the missense codon. The results reported here raise the intriguing possibility that differences in the efficiency of DNA MMR exist between individuals in the human population due to common polymorphisms.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Functional analysis of BRCA1 C-terminal missense mutations identified in breast and ovarian cancer families.

Germline mutations in the breast and ovarian cancer susceptibility gene BRCA1 are responsible for the majority of cases involving hereditary breast and ovarian cancer. Whereas all truncating mutations are considered as functionally deleterious, most of the missense variants identified to date cannot be readily distinguished as either disease-associated mutations or benign polymorphisms. The C-terminal domain of BRCA1 displays an intrinsic transactivation activity, and mutations linked to disease predisposition have been shown to confer loss of such activity in yeast and mammalian cells. In an attempt to clarify the functional importance of the BRCA1 C-terminus as a transcription activator in cancer predisposition, we have characterized the effect of C-terminal germline variants identified in Scandinavian breast and ovarian cancer families. Missense variants A1669S, C1697R, R1699W, R1699Q, A1708E, S1715R and G1738E and a truncating mutation, W1837X, were characterized using yeast- and mammalian-based transcription assays. In addition, four additional missense variants (V1665M, D1692N, S1715N and D1733G) and one in-frame deletion (V1688del) were included in the study. Our findings demonstrate that transactivation activity may reflect a tumor-suppressing function of BRCA1 and further support the role of BRCA1 missense mutations in disease predisposition. We also report a discrepancy between results from yeast- and mammalian-based assays, indicating that it may not be possible to unambiguously characterize variants with the yeast assay alone. We show that transcription-based assays can aid in the characterization of deleterious mutations in the C-terminal part of BRCA1 and may form the basis of a functional assay.

Adult↗
PubMed2003

Identification of CDH1 germline missense mutations associated with functional inactivation of the E-cadherin protein in young gastric cancer probands.

E-cadherin is involved in the formation of cell-junctions and the maintenance of epithelial integrity. Direct evidence of E-cadherin mutations triggering tumorigenesis has come from the finding of inactivating germline mutations of the gene (CDH1) in hereditary diffuse gastric cancer (HDGC). We screened a series of 66 young gastric cancer probands for germline CDH1 mutations, and two novel missense alterations together with an intronic variant were identified. We then analysed the functional significance of the two exonic missense variants found here as well as a third germline missense variant that we previously identified in a HGDC family. cDNAs encoding either the wild-type protein or mutant forms of E-cadherin were stably transfected into CHO (Chinese hamster ovary) E-cadherin-negative cells. Transfected cell-lines were characterized in terms of aggregation, motility and invasion. We show that a proportion of apparently sporadic early-onset diffuse gastric carcinomas are associated with germline alterations of the E-cadherin gene. We also demonstrate that a proportion of missense variants are associated with significant functional consequences, suggesting that our cell model can be used as an adjunct in deciding on the potential pathogenic role of identified E-cadherin germline alterations.

Adult↗
PubMed2003

Aberrant splicing induced by missense mutations in BRCA1: clues from a humanized mouse model.

Numerous missense mutations in human BRCA1 gene have been linked to predisposition to breast cancer. However, the functional significance of the majority of these mutations remains unknown. We have examined the molecular basis for three such cancer-causing mutations. The first mutation, a T-->G transversion in codon 64, is predicted to change a conserved cysteine residue to glycine in the RING finger domain of the 1863 amino acid BRCA1 protein. Using a humanized mouse model we demonstrate that this missense mutation actually results in a functionally null protein. This striking result occurs because the single base alteration generates a new 5' splice site in exon 5 and also disrupts a putative exonic splicing enhancer motif. Consequently, the normal splice donor site is disrupted and an internal cryptic splice site is activated. This results in a 22-nucleotide deletion and the aberrant transcript is predicted to encode a severely truncated protein consisting of only 63 amino acids. To identify other missense mutations in BRCA1 that may result in aberrant splicing, we screened various mutations using the Genscan program. We demonstrate that at least two other missense mutations in codons 1495 and 1823 result in aberrant splicing due to the possible disruption of cis-acting splicing regulatory elements. In conclusion, our study demonstrates for the first time the application of a humanized mouse model for functional analysis of human mutations in mice and also shows the need for a careful examination of the functional consequences of single base alterations and single nucleotide polymorphisms identified in human disease-causing genes.

Alternative Splicing↗
PubMed2005

The establishment of a predictive mutational model of the forkhead domain through the analyses of FOXC2 missense mutations identified in patients with hereditary lymphedema with distichiasis.

The FOX family of transcription factor genes is an evolutionary conserved, yet functionally diverse class of transcription factors that are important for regulation of energy homeostasis, development and oncogenesis. The proteins encoded by FOX genes are characterized by a conserved DNA-binding domain known as the forkhead domain (FHD). To date, disease-causing mutations have been identified in eight human FOX genes. Many of these mutations result in single amino acid substitutions in the FHD. We analyzed the molecular consequences of two disease-causing missense mutations (R121H and S125L) occurring in the FHD of the FOXC2 gene that were identified in patients with hereditary lymphedema with distichiasis (LD) to test the predictive capacity of a FHD structure/function model. On the basis of the FOXC2 solution structure, both FOXC2 missense mutations are located on the DNA-recognition helix of the FHD. A mutation model based on the parologous FOXC1 protein predicts that these FOXC2 missense mutations will impair the DNA-binding and transcriptional activation ability of the FOXC2 protein. When these mutations were analyzed biochemically, we found that both mutations did indeed reduce the DNA binding and transcriptional capacity. In addition, the R121H mutation affected nuclear localization of FOXC2. Together, these data indicate that these FOXC2 missense mutations are functional nulls and that FOXC2 haploinsufficiency underlies hereditary LD and validates the predictive ability of the FOXC1-based FHD mutational model.

Amino Acid Sequence↗
PubMed2004

Human MutL homolog (MLH1) function in DNA mismatch repair: a prospective screen for missense mutations in the ATPase domain.

Germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1 are responsible for the majority of hereditary non-polyposis colorectal cancer (HNPCC), an autosomal-dominant early-onset cancer syndrome. Genetic testing of both MSH2 and MLH1 from individuals suspected of HNPCC has revealed a considerable number of missense codons, which are difficult to classify as either pathogenic mutations or silent polymorphisms. To identify novel MLH1 missense codons that impair MMR activity, a prospective genetic screen in the yeast Saccharomyces cerevisiae was developed. The screen utilized hybrid human-yeast MLH1 genes that encode proteins having regions of the yeast ATPase domain replaced by homologous regions from the human protein. These hybrid MLH1 proteins are functional in MMR in vivo in yeast. Mutagenized MLH1 fragments of the human coding region were synthesized by error-prone PCR and cloned directly in yeast by in vivo gap repair. The resulting yeast colonies, which constitute a library of hybrid MLH1 gene variants, were initially screened by semi-quantitative in vivo MMR assays. The hybrid MLH1 genes were recovered from yeast clones that exhibited a MMR defect and sequenced to identify alterations in the mutagenized region. This investigation identified 117 missense codons that conferred a 2-fold or greater decreased efficiency of MMR in subsequent quantitative MMR assays. Notably, 10 of the identified missense codons were equivalent to codon changes previously observed in the human population and implicated in HNPCC. To investigate the effect of all possible codon alterations at single residues, a comprehensive mutational analysis of human MLH1 codons 43 (lysine-43) and 44 (serine-44) was performed. Several amino acid replacements at each residue were silent, but the majority of substitutions at lysine-43 (14/19) and serine-44 (18/19) reduced the efficiency of MMR. The assembled data identifies amino acid substitutions that disrupt MLH1 structure and/or function, and should assist the interpretation of MLH1 genetic tests.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Classification of BRCA1 missense variants of unknown clinical significance.

BACKGROUND: BRCA1 is a tumour suppressor with pleiotropic actions. Germline mutations in BRCA1 are responsible for a large proportion of breast-ovarian cancer families. Several missense variants have been identified throughout the gene but because of lack of information about their impact on the function of BRCA1, predictive testing is not always informative. Classification of missense variants into deleterious/high risk or neutral/low clinical significance is essential to identify individuals at risk. OBJECTIVE: To investigate a panel of missense variants. METHODS AND RESULTS: The panel was investigated in a comprehensive framework that included (1) a functional assay based on transcription activation; (2) segregation analysis and a method of using incomplete pedigree data to calculate the odds of causality; (3) a method based on interspecific sequence variation. It was shown that the transcriptional activation assay could be used as a test to characterise mutations in the carboxy-terminus region of BRCA1 encompassing residues 1396-1863. Thirteen missense variants (H1402Y, L1407P, H1421Y, S1512I, M1628T, M1628V, T1685I, G1706A, T1720A, A1752P, G1788V, V1809F, and W1837R) were specifically investigated. CONCLUSIONS: While individual classification schemes for BRCA1 alleles still present limitations, a combination of several methods provides a more powerful way of identifying variants that are causally linked to a high risk of breast and ovarian cancer. The framework presented here brings these variants nearer to clinical applicability.

Adolescent↗