A report on the impact of rapid prenatal exome sequencing on the clinical management of 52 ongoing pregnancies: a retrospective review.

OBJECTIVE: Studies have shown that prenatal exome sequencing (PES) improves diagnostic yield in cases of fetal structural malformation. We have retrospectively analysed PES cases from two of the largest fetal medicine centres in the UK to determine the impact of results on management of a pregnancy. DESIGN: A retrospective review of clinical case notes. SETTING: Two tertiary fetal medicine centres. POPULATION: Pregnancies with fetal structural abnormalities referred to clinical genetics via a multidisciplinary team. METHODS: We retrospectively reviewed the notes of all patients who had undergone PES. DNA samples were obtained via chorionic villus sampling or amniocentesis. Variants were filtered using patient-specific panels and interpreted using American College of Medical Genetics guidelines. RESULTS: A molecular diagnosis was made in 42% (18/43) ongoing pregnancies; of this group, there was a significant management implication in 44% (8/18). A positive result contributed to the decision to terminate a pregnancy in 16% (7/43) of cases. A negative result had a significant impact on management in two cases by affirming the decision to continue pregnancy. CONCLUSIONS: We demonstrate that the results of PES can inform pregnancy management. Challenges include variant interpretation with limited phenotype information. These results emphasise the importance of the MDT and collecting phenotype and variant data. As this testing is soon to be widely available, we should look to move beyond diagnostic yield as a measure of the value of PES. TWEETABLE ABSTRACT: Prenatal exome sequencing can aid decision-making in pregnancy management; review ahead of routine implementation in NHS.

Biallelic mutation of MSH2 in primary human cells is associated with sensitivity to irradiation and altered RAD51 foci kinetics.

BACKGROUND: Reports of differential mutagen sensitivity conferred by a defect in the mismatch repair (MMR) pathway are inconsistent in their conclusions. Previous studies have investigated cells established from immortalised human colorectal tumour lines or cells from animal models. METHODS: We examined primary human MSH2-deficient neonatal cells, bearing a biallelic truncating mutation in MSH2, for viability and chromosomal damage after exposure to DNA-damaging agents. RESULTS: MSH2-deficient cells exhibit no response to interstrand DNA cross-linking agents but do show reduced viability in response to irradiation. They also show increased chromosome damage and exhibit altered RAD51 foci kinetics after irradiation exposure, indicating defective homologous recombinational repair. DISCUSSION: The cellular features and sensitivity of MSH2-deficient primary human cells are broadly in agreement with observations of primary murine cells lacking the same gene. The data therefore support the view that the murine model recapitulates early features of MMR deficiency in humans, and implies that the variable data reported for MMR-deficient immortalised human cells may be due to further genetic or epigenetic lesions. We suggest caution in the use of radiotherapy for treatment of malignancies in individuals with functional loss of MSH2.

Posterior fossa malformation in fetuses: a report of 56 further cases and a review of the literature.

OBJECTIVE: The purposes of this study were to determine the outcome of fetuses diagnosed as having a posterior fossa abnormality (PFA) and to find out if there are associated features helpful in determining the prognosis. METHODS: This is a retrospective study of all posterior fossa abnormalities detected prenatally in our Units within the last 10 years. Fifty six patients were selected. Outcome data was collected from the Clinical Genetics Department records and the attending obstetrician or pediatrician. RESULTS: An enlarged cisterna magna (ECM, diameter greater than 10 mm at 18-23 gw) was detected in 22 fetuses, which was isolated in 14 cases. All the patients followed-up (n = 11) with isolated ECM were normal at birth (100%). Non-isolated ECM was present in 8 cases. Further information was available in 7, 5 (71%) of whom had a poor outcome. A Dandy Walker complex abnormality (DWC) was detected in 34 patients. The majority of them had a poor prognosis, 54% if isolated and 84% if non-isolated. CONCLUSIONS: Isolated ECM detected on prenatal scans has a favourable outcome, while DWC is associated with a very high chance of a poor prognosis.

The UK national study of magnetic resonance imaging as a method of screening for breast cancer (MARIBS).

The UK national study of magnetic resonance imaging as a method of screening for breast cancer (MARIBS) is in progress. The study design, accrual to date, and related research projects are described. Revised accrual rates and expected recruitment are given. 15 cancers have been detected to date, from a total of 1236 screening measurements. This event rate and the tumour grades reported are compared with recent reports from other studies in women at high risk of breast cancer.

Prenatal findings on ultrasound and X-ray in a case of overgrowth syndrome associated with increased nuchal translucency.

A case of prenatal diagnosis of an overgrowth syndrome at 30 weeks of gestation is reported. The diagnosis was suggested on the basis of increased fetal growth from 16 weeks onwards, advanced bone age, and characteristic facial features such as hypertelorism, broad forehead and small chin. The fetus presented at 12 weeks with a markedly increased nuchal translucency thickness and generalized skin edema, but normal karyotype. Serial ultrasound scans revealed brain abnormalities including mild unilateral ventriculomegaly and a cyst in the cavum septi pellucidi. The pregnancy was terminated at the parents' request at 32 weeks of gestation and postmortem examination confirmed the prenatal findings. This case demonstrates the possibility of prenatal diagnosis of early overgrowth syndromes and highlights the dilemma arising from the prenatal diagnosis of a non-lethal condition associated with an uncertain prognosis and poorly documented in utero.

Point mutations throughout the GLI3 gene cause Greig cephalopolysyndactyly syndrome.

Greig cephalopolysyndactyly syndrome, characterized by craniofacial and limb anomalies (GCPS; MIM 175700), previously has been demonstrated to be associated with translocations as well as point mutations affecting one allele of the zinc finger gene GLI3. In addition to GCPS, Pallister-Hall syndrome (PHS; MIM 146510) and post-axial polydactyly type A (PAP-A; MIM 174200), two other disorders of human development, are caused by GLI3 mutations. In order to gain more insight into the mutational spectrum associated with a single phenotype, we report here the extension of the GLI3 mutation analysis to 24 new GCPS cases. We report the identification of 15 novel mutations present in one of the patient's GLI3 alleles. The mutations map throughout the coding gene regions. The majority are truncating mutations (nine of 15) that engender prematurely terminated protein products mostly but not exclusively N-terminally to or within the central region encoding the DNA-binding domain. Two missense and two splicing mutations mapping within the zinc finger motifs presumably also interfere with DNA binding. The five mutations identified within the protein regions C-terminal to the zinc fingers putatively affect additional functional properties of GLI3. In cell transfection experiments using fusions of the DNA-binding domain of yeast GAL4 to different segments of GLI3, transactivating capacity was assigned to two adjacent independent domains (TA(1)and TA(2)) in the C-terminal third of GLI3. Since these are the only functional domains affected by three C-terminally truncating mutations, we postulate that GCPS may be due either to haploinsufficiency resulting from the complete loss of one gene copy or to functional haploinsufficiency related to compromised properties of this transcription factor such as DNA binding and transactivation.

Defects in mismatch repair occur after APC mutations in the pathogenesis of sporadic colorectal tumours.

The roles of the intrinsic mutation rate and genomic instability in tumorigenesis are currently controversial. In most colorectal tumours, it is generally supposed that the first mutations occur at the adenomatous polyposis coli (APC) locus; APC mutations are thought to provide cells with a selective advantage but have no known effect on the mutation rate. It has also been suggested that genomic instability is the initiating event in colorectal tumorigenesis and, if this is true, mutations of DNA mismatch repair (MMR) genes (or at similar loci) are the most likely candidates. If defective MMR precedes APC mutations, the APC mutations of colon tumours with defective MMR and hence replication errors (RER+) should differ from those of RER- tumours, in at least three specific ways: (1) a higher frequency of allele loss at APC in RER- tumours; (2) more frameshift than nonsense mutations in RER+ tumours; and (3) APC mutations in simple repeat sequences [(N)n, (N1N2)n, or (N1N2N3)n] in RER+ tumours. We found no evidence that sporadic RER+ and RER- colon cancers (including cell lines) differ in any of these three ways. Although it remains possible that MMR is abnormal in tumours from HNPCC families before APC mutations occur, it is likely that in sporadic colon tumours, APC mutations, rather than genomic instability, are the initiating events in tumorigenesis.

Frequency of germline hereditary non-polyposis colorectal cancer gene mutations in patients with multiple or early onset colorectal adenomas.

BACKGROUND: The hereditary non-polyposis colorectal cancer (HNPCC) syndrome is caused by germline mutations in mismatch repair genes and predisposes individuals to cancers of the colon and other specific sites. On theoretical grounds, it is expected that patients with HNPCC also develop more colorectal adenomas than the general population. In essence, if the mutation rate is raised owing to mutations at a mismatch repair locus, more mutations are expected at loci such as APC (adenomatous polyposis coli) and more adenomas will start to grow. Not all data support this expectation, however. AIM: To search for germline mutations at HNPCC loci in patients with multiple adenomas. SUBJECTS: Twenty five patients (without known APC mutations) who have developed colorectal adenomas in unusually large numbers and, in some cases, at an early age. METHODS: Germline APC mutations were excluded using the protein truction test for exon 15 and mismatch chemical cleavage analysis for remaining exons. Germline HNPCC mutations were detected by using PCR and single strand conformation polymorphism analysis. RESULTS: Just one germline HNPCC mutation was found (4% of cases) and this was of uncertain functional effect. CONCLUSIONS: In general, germline HNPCC mutations are not responsible for the phenotype of patients with multiple colonic adenomas. It is possible that patients with HNPCC tend to develop adenomas more frequently and earlier than the general population, but that this effect is relatively subtle. These results suggest that individuals with colorectal adenomas only should not strictly be classified as "affected" in HNPCC families (although they should certainly not be classified as "unaffected" either and may warrant intensive screening). In the absence of a personal or strong family history of colorectal cancer, it is probably not worthwhile performing diagnostic or predictive genetic testing for HNPCC mutations in subjects with colorectal adenomas. Although undetected APC mutations may be responsible for some of the phenotypes in this sample, as yet uncharacterised adenoma predisposing genes probably exist.

Genetic testing is important in families with a history suggestive of hereditary non-polyposis colorectal cancer even if the Amsterdam criteria are not fulfilled.

BACKGROUND: Clinical screening is still the first-line approach to identification of families with hereditary non-polyposis colorectal cancer (HNPCC). The need for uniformity of diagnosis of the syndrome, particularly in multicentre studies, led to the establishment of a set of minimum diagnostic criteria, the 'Amsterdam criteria'. It is now known that HNPCC is caused by germline defects in the human mismatch repair genes and DNA predictive testing is possible. Defects in two of the known mismatch repair genes, namely hMSH2 and hMLH1, account for over 90 per cent of mutations found in HNPCC families. METHODS: Ten families were identified with pedigrees suggestive of HNPCC (that is with a possible dominant inheritance of HNPCC), but in which the Amsterdam criteria were not fulfilled. Using the technique of single-strand conformational polymorphism analysis, samples were screened from an affected member of each of these ten kindreds for germline mutations in the genes hMSH2 and hMLH1. RESULTS: Mutations were identified in six families. Of these, there were three missense, one nonsense, one frameshift and one putative splice-site mutation. Three of the mutations were in hMSH2 and three in hMLH1. CONCLUSION: This study demonstrates that all families with a pedigree suggestive of HNPCC should be referred to a geneticist even if the Amsterdam criteria are not fulfilled. A knowledge of the gene carrier status enables targeted surveillance and the possibility of early surgical intervention that could be curative.

Use of SSCP analysis to identify germline mutations in HNPCC families fulfilling the Amsterdam criteria.

Hereditary non-polyposis colorectal cancer (HNPCC) is a clinical syndrome characterised by an inherited predisposition to early onset colorectal and uterine cancers and an increased incidence of other cancers. It is caused by germline defects in the human mismatch repair genes. Defects in two of the known mismatch repair genes (namely hMSH2 and hMLH1) account for over 90% of mutations found in HNPCC families. In this study we have identified 14 families that fulfilled the clinical criteria for HNPCC and screened the hMSH2 and hMLH1 genes for germline mutations using single-strand conformational polymorphism (SSCP) analysis and DNA sequencing. Seven mutations were identified. Of these, there were five frameshifts, one missense mutation and a further novel mutation that involved separate transition and transversion changes in successive amino acid residues. Three of the mutations were in hMSH2 and four in hMLH1. The identification of germ-line mutations in an HNPCC family enables targeted surveillance and the possibility of early curative intervention. SSCP is a simple and effective method for identifying most mutations in the human mismatch repair genes using DNA from fresh, frozen or archival material.

Germline HNPCC gene variants have little influence on the risk for sporadic colorectal cancer.

Hereditary non-polyposis colorectal cancer (HNPCC) is a syndrome of inherited bowel and other cancers that has been said to account for up to 15% of all colorectal carcinomas (CRCs). HNPCC can now be diagnosed at the molecular level by detecting germline mutations in genes involved in mismatch repair. A current problem is to determine the prevalence of HNPCC mutations in colon cancer patients with limited or no family history, especially in cases of early onset. We have identified 50 cases of non-polyposis colorectal cancer without a family history of CRC or any other HNPCC cancer, who presented under the age of 45 years. Germline HNPCC variants (at the hMSH2 or hMLH1 loci) were detected in a small minority of cases (6%). The variants that we have found may be new or low penetrance mutations, or even polymorphisms. It remains possible that some of our sample have an inherited predisposition to CRC that is not caused by HNPCC mutations or by known polyposis syndromes. Our data suggest that most HNPCC mutations occur in families and have high or moderate penetrance. New or low penetrance HNPCC mutations probably do not contribute significantly to the risk of colorectal cancer in the general population and probably account for much fewer than 15% of all CRCs. Our results question whether mass population genetic screening programmes are worthwhile for diseases such as HNPCC using current technology.