A novel TRPS1 mutation in a Moroccan family with Tricho-rhino-phalangeal syndrome type III: case report.

BACKGROUND: Tricho-rhino-phalangeal syndrome (TRPS) is an autosomal dominant disorder characterized by craniofacial and skeletal malformations including short stature, thin scalp hair, sparse lateral eyebrows, pear-shaped nose and cone shaped epiphyses. This condition is caused by haploinsufficiency of the TRPS1 gene. Previous genotype-phenotype studies have correlated exon 6 missense mutations with TRPS type III, a severe form of type I with pronounced, facial characteristics, short stature and brachydactyly and differing from type II by the absence of exostoses and mental retardation. CASE PRESENTATION: We report the first case of a Moroccan family, a father and his three children, in which the diagnosis of type III TRPS was suspected based on severe clinical and radiological features. Molecular analysis of the TRPS1 gene revealed a novel missense mutation in exon 6, (p.Ala932Ser), located in the GATA-type DNA-binding zinc finger domain. CONCLUSION: Our observations in this kindred support the previous genotype-phenotype results suggesting that patients with more pronounced facial characteristics and more severe shortening of hands and feet are more likely to have mutation in exon 6 of TRPS1.

Exome sequencing identifies mutations in KIF14 as a novel cause of an autosomal recessive lethal fetal ciliopathy phenotype.

Gene discovery using massively parallel sequencing has focused on phenotypes diagnosed postnatally such as well-characterized syndromes or intellectual disability, but is rarely reported for fetal disorders. We used family-based whole-exome sequencing in order to identify causal variants for a recurrent pattern of an undescribed lethal fetal congenital anomaly syndrome. The clinical signs included intrauterine growth restriction (IUGR), severe microcephaly, renal cystic dysplasia/agenesis and complex brain and genitourinary malformations. The phenotype was compatible with a ciliopathy, but not diagnostic of any known condition. We hypothesized biallelic disruption of a gene leading to a defect related to the primary cilium. We identified novel autosomal recessive truncating mutations in KIF14 that segregated with the phenotype. Mice with autosomal recessive mutations in the same gene have recently been shown to have a strikingly similar phenotype. Genotype-phenotype correlations indicate that the function of KIF14 in cell division and cytokinesis can be linked to a role in primary cilia, supported by previous cellular and model organism studies of proteins that interact with KIF14. We describe the first human phenotype, a novel lethal ciliary disorder, associated with biallelic inactivating mutations in KIF14. KIF14 may also be considered a candidate gene for allelic viable ciliary and/or microcephaly phenotypes.

Mismatch repair deficiency: a temozolomide resistance factor in medulloblastoma cell lines that is uncommon in primary medulloblastoma tumours.

BACKGROUND: Tumours are responsive to temozolomide (TMZ) if they are deficient in O(6)-methylguanine-DNA methyltransferase (MGMT), and mismatch repair (MMR) proficient. METHODS: The effect of TMZ on medulloblastoma (MB) cell killing was analysed with clonogenic survival assays. Expression of DNA repair genes and enzymes was investigated using microarrays, western blot, and immunohistochemistry. DNA sequencing and promoter methylation analysis were employed to investigate the cause of loss of the expression of MMR gene MLH1. RESULTS: Temozolomide exhibited potent cytotoxic activity in D425Med (MGMT deficient, MLH1 proficient; IC(50)=1.7 µM), moderate activity against D341Med (MGMT proficient, MLH1 deficient), and DAOY MB cells (MGMT proficient, MLH1 proficient). MGMT inhibitor O(6)-benzylguanine sensitised DAOY, but not D341Med cells to TMZ. Of 12 MB cell lines, D341Med, D283Med, and 1580WÜ cells exhibited MMR deficiency due to MLH1 promoter hypermethylation. DNA sequencing of these cells provided no evidence for somatic genetic alterations in MLH1. Expression analyses of MMR and MGMT in MB revealed that all patient specimens (n=74; expression array, n=61; immunostaining, n=13) are most likely MMR proficient, whereas some tumours had low MGMT expression levels (according to expression array) or were totally MGMT deficient (3 out of 13 according to immunohistochemistry). CONCLUSION: A subset of MB may respond to TMZ as some patient specimens are MGMT deficient, and tumours appear to be MMR proficient.

Is colorectal surveillance indicated in patients with PTEN mutations?

AIM: Patients with germline phosphatase and tensin homologue (PTEN) mutations develop hamartomatous lesions in several organs and are at increased risk of various malignancies. We assessed the lifetime risk of benign and malignant gastrointestinal lesions in patients with a proven PTEN mutation. METHOD: Data on gender, mutation, dates of birth, last contact, and diagnosis, location and type of gastrointestinal lesions were collected from nine countries. The lifetime risk of gastrointestinal lesions was calculated by Kaplan-Meier methods. RESULTS: A total of 156 patients (67 men, 43%) from 101 families with a PTEN mutation were included. Patients were born between 1928 and 2008. Benign gastrointestinal polyps were reported in 49 (31%) patients at a mean age of 38 years (range 18-62 years) and were most often hamartomas. Twenty-two (44%) patients had upper as well as lower gastrointestinal lesions, 14 (29%) had only colonic lesions and 13 (27%) had gastrointestinal lesions at unknown sites. The cumulative risk of developing benign gastrointestinal polyps was 70% at age 60. Four patients (two men) developed colorectal carcinoma at 53, 57, 59 and 62 years, respectively. The cumulative risk of developing colorectal carcinoma was 18% at age 60. Except for one carcinoid in the small intestine, no upper gastrointestinal cancers were observed. CONCLUSION: Benign gastrointestinal lesions are common in PTEN mutation carriers, and a three- to four-fold increased lifetime risk of colorectal cancer compared with the general population may exist. Colorectal screening of patients with germline PTEN mutations is recommended, starting at age 40 years.

Deletion in Xp22.11: PTCHD1 is a candidate gene for X-linked intellectual disability with or without autism.

Submicroscopic chromosomal anomalies play an important role in the aetiology of intellectual disability (ID) and have been shown to account for up to 10% of non-syndromic forms. We present a family with two affected boys compatible with X-linked inheritance of a phenotype of severe neurodevelopmental disorder co-segregating with a deletion in Xp22.11 exclusively containing the PTCHD1 gene. Although the exact function of this gene is unknown to date, the structural overlap of its encoded patched domain-containing protein 1, the transmembrane protein involved in the sonic hedgehog pathway, and its expression in human cortex and cerebellum as well as in mice and drosophila brain suggests a causative role of its nullisomy in the developmental phenotype of our family. Our findings support the recent notions that PTCHD1 may play a role in X-linked intellectual disability (XLID) and autism disorders.

MutSbeta exceeds MutSalpha in dinucleotide loop repair.

BACKGROUND: The target substrates of DNA mismatch recognising factors MutSalpha (MSH2+MSH6) and MutSbeta (MSH2+MSH3) have already been widely researched. However, the extent of their functional redundancy and clinical substance remains unclear. Mismatch repair (MMR)-deficient tumours are strongly associated with microsatellite instability (MSI) and the degree and type of MSI seem to be dependent on the MMR gene affected, and is linked to its substrate specificities. Deficiency in MSH2 and MSH6 is associated with both mononucleotide and dinucleotide repeat instability. Although no pathogenic MSH3 mutations have been reported, its deficiency is also suggested to cause low dinucleotide repeat instability. METHODS: To assess the substrate specificities and functionality of MutSalpha and MutSbeta we performed an in vitro MMR assay using three substrate constructs, GT mismatch, 1 and 2 nucleotide insertion/deletion loops (IDLs) in three different cell lines. RESULTS: Our results show that though MutSalpha alone seems to be responsible for GT and IDL1 repair, MutSalpha and MutSbeta indeed have functional redundancy in IDL2 repair and in contrast with earlier studies, MutSbeta seems to exceed MutSalpha. CONCLUSION: The finding is clinically relevant because the strong role of MutSbeta in IDL2 repair indicates MSH3 deficiency in tumours with low dinucleotide and no mononucleotide repeat instability.

Prognostic and predictive value of TOPK stratified by KRAS and BRAF gene alterations in sporadic, hereditary and metastatic colorectal cancer patients.

BACKGROUND: Our aim was to investigate the prognostic and predictive value of the oncogenic MAPKK-like protein T-cell-originated protein kinase (TOPK) stratified by KRAS and BRAF mutations in patients with sporadic, hereditary and metastatic colorectal cancer (CRC) treated with anti-EGFR therapy. METHODS: Immunohistochemistry (IHC) for TOPK was performed on four study groups. Group 1 included two subgroups of 543 and 501 sporadic CRC patients used to test the reliability of TOPK expression by IHC. In Group 2, representing an additional 222 sporadic CRCs, the prognostic effect of TOPK stratified by KRAS and BRAF was assessed. The prognostic effect of TOPK was further analysed in Group 3, representing 71 hereditary Lynch syndrome-associated CRC patients. In Group 4, the predictive and prognostic value of TOPK was analysed on 45 metastatic patients treated with cetuximab or panitumumab stratified by KRAS and BRAF gene status. RESULTS: In both sporadic CRC subgroups (Group 1), associations of diffuse TOPK expression with clinicopathological features were reproducible. Molecular analysis of sporadic CRCs in Group 2 showed that diffuse TOPK expression was associated with KRAS and BRAF mutations (p<0.001) and with poor outcome in patients with either mutation in univariate and multivariate analysis (P=0.017). In hereditary patients (Group 3), diffuse TOPK was linked to advanced pT stage. In metastatic patients treated with anti-EGFR therapy (Group 4), diffuse TOPK expression was linked to dismal outcome despite objective response to treatment (P=0.01). CONCLUSIONS: TOPK expression is an unfavourable prognostic indicator in sporadic patients with KRAS or BRAF mutations and also in patients with metastatic disease experiencing a response to anti-EGFR therapies. The inhibition of TOPK, which could benefit 30-40% of CRC patients, may represent a new avenue of investigation for targeted therapy.

Attenuated familial adenomatous polyposis: results from an international collaborative study.

AIM: The study aimed to describe genetical and clinical features of attenuated familial adenomatous polyposis (AFAP) and to propose clinical criteria and guidelines for treatment and surveillance. METHOD: A questionnaire study was carried out of polyposis registries with data on patients with presumed AFAP, defined as having ≤ 100 colorectal adenomas at age ≥ 25. RESULTS: One hundred and ninety-six patients were included. The median number of adenomas was 25 (0-100) with a uniform distribution of colorectal adenomas and carcinomas (CRC). Age at CRC diagnosis was delayed by 15 years compared with classic FAP. Eighty-two patients had a colectomy and an ileorectal anastomosis and 5/82 (6%) had a secondary proctectomy. The location of the mutation in the APC gene was known in 69/171 (40%) tested patients. Only 15/29 (52%) of mutations in APC were found in parts of the gene usually associated with AFAP (the 5' end, exon 9 and 3' end). CONCLUSIONS: A subset of FAP patients with a milder phenotype does exist and treatment and surveillance had to be modified accordingly. The mutation detection rate is lower than in classic FAP and mutations in AFAP patients are located throughout the APC gene. We propose the following clinical diagnostic criteria for AFAP: a dominant mode of inheritance of colorectal adenomatosis and <100 colorectal adenomas at age 25 or older. Colonoscopy had to be preferred to sigmoidoscopy and surveillance had to be life-long. In the majority of patients, prophylactic colectomy and ileorectal anastomosis are recommended at the age of 20-25 years.

LAMA2 gene analysis in a cohort of 26 congenital muscular dystrophy patients.

Congenital muscular dystrophy type 1A (MDC1A) is caused by mutations in the LAMA2 gene encoding laminin-alpha2. We describe the molecular study of 26 patients with clinical presentation, magnetic resonance imaging and/or laminin-alpha2 expression in muscle, compatible with MDC1A. The combination of full genomic sequencing and complementary DNA analysis led to the particularly high mutation detection rate of 96% (50/52 disease alleles). Besides 22 undocumented polymorphisms, 18 different mutations were identified in the course of this work, 14 of which were novel. In particular, we describe the first fully characterized gross deletion in the LAMA2 gene, encompassing exon 56 (c.7750-1713_7899-2153del), detected in 31% of the patients. The only two missense mutations detected were found in heterozygosity with nonsense or truncating mutations in the two patients with the milder clinical presentation and a partial reduction in muscle laminin-alpha2. Our results corroborate the previous few genotype/phenotype correlations in MDC1A and illustrate the importance of screening for gross rearrangements in the LAMA2 gene, which may be underestimated in the literature.

Cystic fibrosis and intrauterine death.

Fetal death is not commonly associated with cystic fibrosis (CF). We report a case of late intrauterine death attributed to cardiovascular failure and shock consequent to malrotation and intestinal volvulus in a fetus affected with CF. An argument is made that CF promoted this deleterious incident. Whole blood or cell-rich tissue specimens should be preserved and genetic testing for CF considered in stillbirths with intestinal complications.

Disease severity and genetic pathways in attenuated familial adenomatous polyposis vary greatly but depend on the site of the germline mutation.

BACKGROUND: Attenuated familial adenomatous polyposis (AFAP) is associated with germline mutations in the 5', 3', and exon 9 of the adenomatous polyposis coli (APC) gene. These mutations probably encode a limited amount of functional APC protein. METHODS AND RESULTS: We found that colonic polyp number varied greatly among AFAP patients but members of the same family tended to have more similar disease severity. 5' Mutants generally had more polyps than other patients. We analysed somatic APC mutations/loss of heterozygosity (LOH) in 235 tumours from 35 patients (16 families) with a variety of AFAP associated germline mutations. In common with two previous studies of individual kindreds, we found biallelic changes ("third hits") in some polyps. We found that the "third hit" probably initiated tumorigenesis. Somatic mutation spectra were similar in 5' and 3' mutant patients, often resembling classical FAP. In exon 9 mutants, in contrast, "third hits" were more common. Most "third hits" left three 20 amino acid repeats (20AARs) on the germline mutant APC allele, with LOH (or proximal somatic mutation) of the wild-type allele; but some polyps had loss of the germline mutant with mutation leaving one 20AAR on the wild-type allele. CONCLUSIONS: We propose that mutations, such as nt4661insA, that leave three 20AARs are preferentially selected in cis with some AFAP mutations because the residual protein function is near optimal for tumorigenesis. Not all AFAP polyps appear to need "three hits" however. AFAP is phenotypically and genetically heterogeneous. In addition to effects of different germline mutations, modifier genes may be acting on the AFAP phenotype, perhaps influencing the quantity of functional protein produced by the germline mutant allele.

N-acetyltransferase 2 influences cancer prevalence in hMLH1/hMSH2 mutation carriers.

Hereditary nonpolyposis colorectal cancer (HNPCC), an inherited cancer predisposition syndrome, has been associated with germline mutations in DNA mismatch repair (MMR) genes. Because a deficiency in MMR does not predict a specific cancer phenotype, modifying genes may account in part for the variation in disease expression. We determined the N-acetyltransferase 2 (NAT2) genotype in 26 unaffected and 52 cancer-affected hMLH1/hMSH2 mutation carriers coming from 21 Swiss HNPCC families. Slow acetylators were found to be significantly (P < 0.03) more prevalent in the group of affected mutation carriers. Our results suggest a protective effect of the NAT2 rapid acetylator phenotype, an observation that could have implications for genetic counseling and management of MMR gene mutation carriers.

Nontruncating APC germ-line mutations and mismatch repair deficiency play a minor role in APC mutation-negative polyposis.

Familial adenomatous polyposis, an autosomal-dominantly inherited colorectal cancer predisposition syndrome, is caused by germ-line mutations in the adenomatous polyposis coli (APC) gene. Despite the use of different screening methods, studies worldwide fail to identify APC mutations in 20-50% of all familial adenomatous polyposis patients (APC mutation-negatives). In this study, missense mutations in the coding region of the APC gene, which would have been missed by the protein truncation test, as well as mutations in the APC promoter and the 3' untranslated region, were determined by the single nucleotide polymorphism discovery assay and direct DNA sequencing in 31 mutation-negative polyposis patients. Seventeen gene alterations were identified, whereof four (12.9%) represent possibly pathogenic germ-line mutations: silent A290T (promoter) and A8822G (3' untranslated region) as well as missense R99W and E1317Q (coding region). The 27 remaining, truly APC mutation-negative polyposis patients displayed a significantly later age at diagnosis compared with APC mutation carriers (46.1 versus 35.2 years; P < 0.01). APC mutation-negative individuals with >100 colonic polyps were more likely to present with extracolonic disease (P < 0.05) than those with <100. Assessment of microsatellite instability (MSI), a hallmark of mismatch repair deficiency, in 68 tumors from 21 truly APC mutation-negative patients, identified 4 (5.9%) unstable tubulo-villous adenomas (3 MSI-High and 1 MSI-Low), stemming from 4 (19%) unrelated individuals and likely to be caused by hMLH1 promoter hypermethylation. In conclusion, only a small proportion of APC germ-line mutation carriers is missed by the protein truncation test, and mismatch repair deficiency does not seem to substantially contribute to tumor development in APC mutation-negative polyposis patients.

An MLH1 haplotype is over-represented on chromosomes carrying an HNPCC predisposing mutation in MLH1.

BACKGROUND: The mismatch repair gene, MLH1, appears to occur as two main haplotypes at least in white populations. These are referred to as A and G types with reference to the A/G polymorphism at IVS14-19. On the basis of preliminary experimental data, we hypothesised that deviations from the expected frequency of these two haplotypes could exist in carriers of disease associated MLH1 germline mutations. METHODS: We assembled a series (n=119) of germline MLH1 mutation carriers in whom phase between the haplotype and the mutation had been conclusively established. Controls, without cancer, were obtained from each contributing centre. Cases and controls were genotyped for the polymorphism in IVS14. RESULTS: Overall, 66 of 119 MLH1 mutations occurred on a G haplotype (55.5%), compared with 315 G haplotypes on 804 control chromosomes (39.2%, p=0.001). The odds ratio (OR) of a mutation occurring on a G rather than an A haplotype was 1.93 (95% CI 1.29 to 2.91). When we compared the haplotype frequencies in mutation bearing chromosomes carried by people of different nationalities with those seen in pooled controls, all groups showed a ratio of A/G haplotypes that was skewed towards G, except the Dutch group. On further analysis of the type of each mutation, it was notable that, compared with control frequencies, deletion and substitution mutations were preferentially represented on the G haplotype (p=0.003 and 0.005, respectively). CONCLUSION: We have found that disease associated mutations in MLH1 appear to occur more often on one of only two known ancient haplotypes. The underlying reason for this observation is obscure, but it is tempting to suggest a possible role of either distant regulatory sequences or of chromatin structure influencing access to DNA sequence. Alternatively, differential behaviour of otherwise similar haplotypes should be considered as prime areas for further study.

Exclusion of an extracolonic disease modifier locus on chromosome 1p33-36 in a large Swiss familial adenomatous polyposis kindred.

Familial adenomatous polyposis (FAP), an autosomal dominantly inherited colorectal cancer predisposition syndrome, displays considerable inter- and intrafamilial phenotypic heterogeneity, which represents a major problem in genetic counselling of APC mutation carriers. The Min mouse model indicated a putative disease modifier locus on chromosome 4, which is syntenic to human chromosome 1p35-36. This finding was subsequently supported by parametric and nonparametric linkage analyses in FAP families, however, without identifying functional variants in candidate genes. Recently, germline mutations in the base-excision repair gene MYH (1p33-34) have been described in patients with multiple adenomas, pointing to a possible role as disease modifier in FAP. Here, we present critical reassessment of one of the largest FAP kindreds published, which was previously used in linkage mapping of 1p35-36. In this family, all affected members harbour the same APC germline mutation (5945delA), but display marked phenotypic variability, in particular regarding the occurrence of extracolonic disease that segregates in several branches of the family tree. Using updated clinical information, additional mutation carriers and polymorphic markers, fine mapping of the critical region as well as mutation analysis of the MYH gene were performed. These investigations allowed us to significantly exclude (i) the 1p33-36 region as a modifier locus and (ii) MYH as a modifier gene for extracolonic disease in this FAP kindred. Our results do not eliminate 1p33-36 from suspicion in other families, but clearly indicate that in our family linkage analysis of further putative candidate regions is necessary to identify a disease modifier locus in FAP.

Association of extracolonic manifestations of familial adenomatous polyposis with acetylation phenotype in a large FAP kindred.

Familial adenomatous polyposis coli (FAP) has been shown to be associated with germline mutations of the adenomatous polyposis gene (APC) on chromosome 5. Extra-colonic manifestations also occur in FAP and include desmoid tumors, epidermoid cysts and osteomas. The combination of FAP with extracolonic symptoms is commonly referred to as Gardner's syndrome. It remains difficult, however, to predict which patients may have a propensity to develop extracolonic manifestations. The rapid acetylation phenotype is believed to be associated with an increased likelihood of sporadic colorectal cancer, whereas the slow acetylation phenotype is recognized as a predisposing factor for bladder cancer. The slow acetylation phenotype is caused by mutant alleles of the cytosolic enzyme N-acetyltransferase (NAT2). In this study, we determined the NAT2 genotype in members of one large FAP family and three smaller ones all of which had been shown to harbor the same germline APC gene mutation. We observed a significant correlation between slow acetylation genotypes and extracolonic manifestations of the disease. Rapid acetylation genotypes were not overrepresented in colorectal cancer cases in this family as compared to the frequency of this genotype in the normal Caucasian population.

Genetics of hereditary colon cancer - a basis for prevention?

Hereditary colorectal cancer syndromes are among the best in vivo models to study colorectal carcinogenesis and the influence of putative modifiers of the cancer risk. The present knowledge regarding the wide range of colorectal cancer (CRC) susceptibilities and the histological and molecular changes they elicit is leading to a very dynamic and integrated concept of tumorigenesis in the colon and to new views about prevention and early treatment of cancer.

[Genetic testing in pregnancy]. / Genetische Untersuchungen während der Schwangerschaft.

First trimester risk screening is probably the major methodological advance in identifying pregnancies at increased risk for genetic disease during recent years with an impact on all pregnancies. The high detection rate with moderate false positive rates will reduce the over-all number of invasive procedures as compared to the traditional approach based on maternal age exclusively, in particular considering the demographic shift towards higher mean maternal age. Non-invasive prenatal diagnosis from fetal cells or DNA in the maternal circulation remains an experimental approach, despite a growing number of reports on successful diagnoses of single gene disorders. In the lab molecular cytogenetic approaches have considerably broadened the diagnostic spectrum of conventional karyotyping and facilitated a rapid diagnosis of selected frequent aneuploidies. Molecular genetic testing, in particular on chorionic villi, allows an early and reliable diagnosis of a growing number of severe monogenic conditions. A restrictive legislation has hampered the development of preimplantation genetic diagnosis in German speaking countries, only a few groups work on polar body diagnosis, a legal but restricted alternative.