Missense mutations in hMLH1 and hMSH2 are associated with exonic splicing enhancers

There is a critical need to understand why missense mutations are deleterious. The deleterious effects of missense mutations are commonly attributed to their impact on primary amino acid sequence and protein structure. However, several recent studies have shown that some missense mutations are deleterious because they disturb cis-acting splicing elements-so-called "exonic splicing enhancers" (ESEs). It is not clear whether the ESE-related deleterious effects of missense mutations are common. We have evaluated colocalization of pathogenic missense mutations (found in affected individuals) with high-score ESE motifs in the human mismatch-repair genes hMSH2 and hMLH1. We found that pathogenic missense mutations in the hMSH2 and hMLH1 genes are located in ESE sites significantly more frequently than expected. Pathogenic missense mutations also tended to decrease ESE scores, thus leading to a higher propensity for splicing defects. In contrast, nonpathogenic missense mutations (polymorphisms found in unaffected individuals) and nonsense mutations are distributed randomly in relation to ESE sites. Comparison of the observed and expected frequencies of missense mutations in ESE sites shows that pathogenic effects of >/=20% of mutations in hMSH2 result from disruption of ESE sites and disturbed splicing. Similarly, pathogenic effects of >/=16% of missense mutations in the hMLH1 gene are ESE related. The colocalization of pathogenic missense mutations with ESE sites strongly suggests that their pathogenic effects are splicing related.     

Missense mutations in CRELD1 are associated with cardiac atrioventricular septal defects

Atrioventricular septal defects (AVSD) are common cardiovascular malformations, occurring in 3.5/10,000 births. Although frequently associated with trisomy 21, autosomal dominant AVSD has also been described. Recently we identified and characterized the cell adhesion molecule CRELD1 (previously known as "cirrin") as a candidate gene for the AVSD2 locus mapping to chromosome 3p25. Analysis of the CRELD1 gene from individuals with non-trisomy 21-associated AVSD identified heterozygous missense mutations in nearly 6% of this population, including mutations in isolated AVSD and AVSD associated with heterotaxy syndrome. CRELD1 is the first human gene to be implicated in the pathogenesis of isolated AVSD and AVSD in the context of heterotaxy, which provides an important step in unraveling the pathogenesis of AVSD.     

Missense mutations in the BMP15 gene are associated with ovarian failure

Premature ovarian failure (POF) is an unexplained amenorrhoea (>6 months) with raised levels of gonadotropins (FSH>40 U/L) occurring before the age of 40 years. Recent studies have elucidated the role of oocyte derived growth factors (BMP15 and GDF9) in maintenance of folliculogenesis, granulosa cell (GC) proliferation and overall fertility. Our recently published work showed presence of two rare missense variants in the GDF9 gene associated with ovarian failure (Dixit et al. 2005, Menopause 12:749–754). The present case-control study has been structured to establish the role of BMP15 germline status associated with ovarian failure. Sequence analysis of the coding region of the BMP15 gene was carried out in a cohort of women with POF (n=133), primary amenorrhoea (n=60), and secondary amenorrhoea (n=9) compared with control females (n=197). This study revealed a total of 18 germline variants in the coding region of BMP15 gene, including 16 novel variants. These novel variants include one intronic variant, one 3’ flanking variant, one silent variant, and 13 missense variants. Eleven missense variants were present only in cases with complete absence in the control females. The remaining two missense variants viz. c.308A>G (p.Asn103Ser) and c.788_789insTCT (p.Leu263_Arg264insLeu) were present both in the cases and in the controls. The c.788_789insTCT variant was significantly higher in primary amenorrhoea cases than in the controls (Fisher’s exact test, P=0.034). Three frequent variants c.-9C>G, c.308A>G, and c.852C>T were chosen for haplotyping. The haplotype G-G-C was found to be significantly associated with ovarian failure (P=0.0075). In a nutshell, the BMP15 gene is highly associated with etiology of ovarian failure.     

GAEC1 mutations and copy number aberration is associated with biological aggressiveness of colorectal cancer

GAEC1 (gene amplified in oesophageal cancer 1) is a transforming oncogene with tumorigenic potential observed in both oesophageal squamous cell carcinoma and colorectal cancer. Nonetheless, there has been a lack of study done on this gene to understand how this gene exert its oncogenic properties in cancer. This study aims to identify novel mutation sites in GAEC1. To do so, seventy-nine matched colorectal cancers were tested for GAEC1 mutation via Sanger sequencing. The mutations noted were investigated for the correlations with the clinicopathological parameters of the patients with the cancer. Additionally, GAEC1 copy number aberration (CNA), mRNA and protein expression were determined with the use of droplet digital (dd) polymerase chain reaction (PCR), real-time PCR and Western blot (confirmed with immunofluorescence analysis). GAEC1 mutation was noted in 8.8% (n?=?7/79) of the cancer tissues including one missense mutation, four loss of heterozygosity (LOH) and two substitutions. These mutations were significantly associated with cancer perforation (p?=?0.021). GAEC1 mutation is frequently associated with increased GAEC1 protein expression. Nevertheless, GAEC1 mRNA and protein are only weakly associated. Taken together, GAEC1 mutation affects GAEC1 expression and is associated with poorer clinical outcomes. This further strengthens the role of GAEC1 as an oncogene.     

Enhanced detection of deleterious and other germline mutations of hMSH2 and hMLH1 in Japanese hereditary nonpolyposis colorectal cancer kindreds

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal, dominantly inherited cancer-prone syndrome. Here, we describe a novel and efficient approach for screening mutations of two major HNPCC susceptibility genes, hMSH2 and hMLH1. The system consists of RNA extraction from whole blood treated with the translation inhibitor, followed by long RT-PCR of the entire coding regions combined with direct sequencing. In analysis of 15 kindreds suspicious for HNPCC, 8 samples were subjected to analysis after puromycin treatment and 7 samples were analyzed without puromycin treatment. Three deleterious mutations were detected in the kindreds with puromycin treatment, while none were observed in those without puromycin. Signals from mutated alleles were enhanced after puromycin treatment and easily distinguished from the wild-type allele, achieved by suppression of nonsense-mediated mRNA decay. Furthermore, 12 other mutations were detected in 15 kindreds. The system is considered to be a reliable and useful approach for detecting germline mutations of hMSH2 and hMLH1 in HNPCC kindreds.     

Missense mutations associated with RFLP haplotypes 1 and 4 of the human phenylalanine hydroxylase gene.

We report missense mutations associated with haplotype 1 and haplotype 4 alleles of the human phenylalanine hydroxylase (PAH) gene. Individual exon-containing regions were amplified by polymerase chain reaction from genomic DNA of a PKU patient who was a haplotype 1/4 compound heterozygote. The amplified DNA fragments were subcloned into M13 for sequence analysis. Missense mutations were observed in exons 5 and 7, resulting in the substitution of Arg by Gln at residues 158 and 261 of the enzyme, respectively. Expression analysis in heterozygous mammalian cells after site-directed mutagenesis demonstrated that the Arg158-to-Gln158 mutation is a PKU mutation, whereas the Arg261-to-Gln261 mutation is apparently silent in the assay system. Hybridization analysis using allele-specific oligonucleotide probes demonstrated that the Arg158-to-Gln158 mutation is present in two of six mutant haplotype 4 alleles among the Swiss and constitutes about 40% of all mutant haplotype 4 alleles in the European population. The mutation is not present in normal alleles or in any mutant alleles of other haplotypes. The results provide conclusive evidence that there is linkage disequilibrium between mutation and haplotype in the PAH gene and that multiple mutations have occurred in the PAH gene of a prevalent haplotype among Caucasians.     

Majority of hMLH1 mutations responsible for hereditary nonpolyposis colorectal cancer cluster at the exonic region 15-16.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer susceptibility condition. Inherited mutations in at least four DNA mismatch repair genes, hMSH2, hMLH1, hPMS1, and hPMS2, are known to cause HNPCC. In this study we used denaturing gradient gel electrophoresis (DGGE) to screen for hMLH1 mutations in 34 unrelated HNPCC families (30 Dutch, 3 Italian, and 1 Danish). Ten novel pathogenic germ-line mutations (seven affecting splice sites, two frameshifts, and one in-frame deletion of a single amino acid) have been identified in 12 (35%) of these families. In a previous study, hMSH2 mutations were found in 21% of the same families. While the spectrum of mutations at the hMSH2 gene among HNPCC patients appears heterogeneous, a cluster of hMLH1 mutations has been found in the region encompassing exons 15 and 16, which accounts for 50% of all the independent hMLH1 mutations described to date and for > 20% of the unrelated HNPCC kindreds here analyzed. This unexpected finding has a great practical value in the clinical scenario of genetic services.     

Mutation analysis of hMSH2 and hMLH1 in colorectal cancer patients in India

The aim of this work was to study the mutation profile in hMSH2 and hMLH1 genes in hereditary nonpolyposis colorectal cancer (HNPCC) patients in India. On the basis of the Bethesda criteria, 31 colorectal cancer patients were studied first for microsatellite instability, using the five markers recommended by the Bethesda guidelines. Twelve of 31 tumor samples were found to be MSI-H, 9 of 31 were MSI-L, and the rest were MSS. The 12 patients with MSI-H were analyzed for mutations in hMSH2 and hMLH1 genes using PCR-denaturing high-performance liquid chromatography (dHPLC), followed by sequencing of samples showing abnormal peaks. Of the five mutations detected, three were found to be deleterious mutations (hMSH2-R680X, hMLH1-E671X, and a splice junction mutation IVS16-2A --> G); one had a mutation of probable significance (hMLH1-C680G) and one was of unknown significance (hMSH2-R171K). This study has also shown that most of the early-onset colon (4/7) and early-onset rectal (15/21) cancers are MSS or MSI-L. This is the first study to describe the mutation in hMSH2 and hMLH1 in Indian patients, a low incidence region for colorectal cancer. A two-stage procedure using MSI testing followed by PCR-dHPLC was found to be an efficient method in studying the mutation profile in high-risk patients.     

Reduced frequency of extracolonic cancers in hereditary nonpolyposis colorectal cancer families with monoallelic hMLH1 expression.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant disease caused by mutations in one of at least four different DNA mismatch repair genes, hMLH1, hMSH2, hPMS1, and hPMS2. Phenotypically, HNPCC is characterized by the early onset of colorectal cancers and various extracolonic cancers. Depending on the presence or absence of extracolonic tumors, HNPCG-has been divided into two syndromes (Lynch syndrome I and Lynch syndrome II), but, so far, no correlation to distinct genotypes has been demonstrated. In this study, we present a frequent hMLH1 intron 14 founder mutation that is associated with a highly reduced frequency of extracolonic tumors. The mutation disrupts the splice donor site and silences the mutated allele. Tumors exhibited microsatellite instability, and loss of the wild-type hMLH1 allele was prevalent. We propose that the mutation results in a milder phenotype, because the mutated hMLH1 protein is prevented from exerting a dominant negative effect on the concerted action of the mismatch repair system.     

Prevalence of germline mutations of hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 genes in 75 French kindreds with nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is a syndrome characterized by familial predisposition to colorectal carcinoma and extracolonic cancers of the gastrointestinal, urological, and female reproductive tracts. This dominant disorder is caused by germline defects in one of at least five DNA mismatch repair (MMR) genes: hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 (GTBP). Germline mutations of hMSH2 and hMLH1 are also frequently identified in families not fulfilling all the Amsterdam criteria, thereby demonstrating that the involvement of these genes is not confined to typical HNPCC. To evaluate the respective involvement of the various MMR genes in typical and incomplete HNPCC syndromes, we have performed an analysis of the hMLH1, hMSH2, hPMS1, hPMS2, and hMSH6 genes in a large series of French kindreds (n=75) with colorectal tumors and/or aggregation of extracolonic cancers belonging to the HNPCC spectrum. Mutational analysis has been performed in all families, without preselection for the tumor phenotype. We have detected 26 pathogenic germline mutations of the hMLH1 and hMSH2 genes and several novel variants of the hPMS1, hPMS2, and hMSH6 genes. Our data confirm that, regardless of the type of families and the tumor phenotype, hPMS1, hPMS2, and hMSH6 germline mutations are rare in familial aggregation of colorectal cancers. Furthermore, they suggest that the presence of multiple primary malignancies in a single individual and the observation of extracolonic tumors in relatives of a colorectal cancer patient should be included among the guidelines for referring patients for genetic testing. Electronic Publication     

Germline PKHD1 mutations are protective against colorectal cancer

The autosomal recessive polycystic kidney disease (ARPKD) gene, PKHD1, has been implicated in the genesis or growth of colorectal adenocarcinoma, as a high level of somatic mutations was found in colorectal tumor tissue. To determine whether carriers of a single PKHD1 mutation are at increased risk of colorectal carcinoma, we assessed the prevalence of the commonest European mutation, T36M. First, we assayed a European cohort of ARPKD patients and found T36M was responsible for 13.1% of mutations. We then investigated two European cohorts with colorectal adenocarcinoma versus two control cohorts of similar age and gender. Screening for the most common PKHD1 mutation, T36M, we detected 15:3,603 (0.42%) controls versus 1:3,767 (0.027%) colorectal cancer individuals, indicating that heterozygous PKHD1 mutations are not a risk factor and are protective (p=0.0002). We also show that the carriage rate for PKHD1 mutations in the European population is higher than previous accepted at 3.2% (1:31 genomes).     

The interplay between hMLH1 and hMRE11: role in MMR and the effect of hMLH1 mutations

Our previous studies indicate that hMRE11 plays a role in MMR, and this function of hMRE11 is most likely mediated by the hMLH1-hMRE11 interaction. Here, we explored the functional implications of the hMLH1-hMRE11 interaction in MMR and the effects of hMLH1 mutations on their interaction. Our in vitro MMR assay demonstrated that the dominant-negative hMRE11^452-634 mutant peptide (i.e., harboring only the hMLH1-interacting domain) imparted a significant reduction in both 3′ excision and 3′-directed MMR activities. Furthermore, the expression of hMRE11^452-634, and to a lesser extent hMRE11^1-634 (ATLD1), impaired G2/M checkpoint control in response to MNU and cisplatin treatments, rendering cells resistant to killings by these two anticancer drugs. Analysis of 38 hMLH1 missense mutations showed that the majority of mutations caused significant (>50%) reductions in their interaction with hMRE11, suggesting a potential link between aberrant protein interaction and the pathogenic effects of hMLH1 variants.     

Missense mutations in SGLT1 cause glucose-galactose malabsorption by trafficking defects

Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder caused by defects in the Na+/glucose cotransporter (SGLT1). Neonates present with severe diarrhea while on any diet containing glucose and/or galactose [1]. This study focuses on a patient of Swiss and Dominican descent. All 15 exons of SGLT1 were screened using single stranded conformational polymorphism analyses, and aberrant PCR products were sequenced. Two missense mutations, Gly318Arg and Ala468Val, were identified. SGLT1 mutants were expressed in Xenopus laevis oocytes for radiotracer uptake, electrophysiological experiments, and Western blotting. Uptakes of [14C]alpha-methyl-d-glucoside by the mutants were 5% or less than that of wild-type. Two-electrode voltage-clamp experiments confirmed the transport defects, as no noticeable sugar-induced current could be elicited from either mutant [2]. Western blots of cell protein showed levels of each SGLT1 mutant protein comparable to that of wild-type, and that both were core-glycosylated. Presteady-state current measurements indicated an absence of SGLT1 in the plasma membrane. We suggest that the compound heterozygote missense mutations G318R and A468V lead to GGM in this patient by defective trafficking of mutant proteins from the endoplasmic reticulum to the plasma membrane.     

Missense mutations in the catalase-peroxidase gene, katG, are associated with isoniazid resistance in Mycobacterium tuberculosis

The toxicity of the powerful anti-tuberculosis drug isoniazid (INH) is believed to be mediated by the haem-containing enzyme catalase-peroxidase, encoded by the katG gene of Mycobacterium tuberculosis. Compelling evidence for this was obtained by studying a panel of INH-resistant clinical isolates using a novel strategy based on the polymerase chain reaction and single-strand-conformation polymorphism analysis (PCR-SSCP) to detect mutations in katG. In most cases INH resistance was associated with missense mutations while in a small number of strains the gene had been completely, or partially, deleted. The missense mutations fell into two groups, the larger of which contained several independent mutations that affected the N-terminal peroxidase domain of the protein, resulting in the production of a catalase peroxidase with strongly reduced enzyme activity and increased heat lability. The effects of these substitutions could be interpreted by means of molecular modelling using the crystal structure of the related enzyme cytochrome c peroxidase from yeast as a template. The second group comprises a frequently occurring amino acid substitution and a single mutation that are both located in the C-terminal domain but do not noticeably alter either enzyme activity or heat stability.