ACMG statement on noninvasive prenatal screening for fetal aneuploidy.

Noninvasive assessment of the fetal genome is now possible using next-generation sequencing technologies. The isolation of fetal DNA fragments from maternal circulation in sufficient quantity and sizes, together with proprietary bioinformatics tools, now allows patients the option of noninvasive fetal aneuploidy screening. However, obstetric care providers must become familiar with the advantages and disadvantages of the utilization of this approach as analysis of cell-free fetal DNA moves into clinical practice. Once informed, clinicians can provide efficient pretest and posttest counseling with the goal of avoiding patient harm. It is in the public's best interest that test results contain key elements and that laboratories adhere to established quality control and proficiency testing standards. The analysis of cell-free fetal DNA in maternal circulation for fetal aneuploidy screening is likely the first of major steps toward the eventual application of whole fetal genome/whole fetal exome sequencing.

Plasma IL-6, its soluble receptors and F2-isoprostanes at rest and during exercise in chronic fatigue syndrome.

The aim of the current study was to investigate the levels of interleukin-6 (IL-6), its soluble receptors (sIL-6R and sgp130) and F(2)-isoprostanes, at rest and during exercise, in patients with chronic fatigue syndrome (CFS). Six male CFS patients and six healthy controls performed an incremental exercise test to exhaustion and a submaximal exercise bout to exhaustion. Blood samples taken in the submaximal test at rest, immediately post-exercise and 24 h post-exercise were analyzed for IL-6, sIL-6R, sgp130 and F(2)-isoprostanes. A further 33 CFS and 33 healthy control participants gave a resting blood sample for IL-6 and sIL-6R measurement. During the incremental exercise test only power output at the lactate threshold was lower (P<0.05) in the CFS group. F(2)-isoprostanes were higher (P<0.05) in CFS patients at rest and this difference persisted immediately and 24 h post-exercise. The exercise study found no differences in IL-6, sIL-6R or sgp130 at any time point between groups. In the larger resting group, there were no differences in IL-6 and sIL-6R between CFS and control groups. This investigation has demonstrated that patients with CFS do not have altered plasma levels of IL-6, sIL-6R or sgp130 either at rest or following exercise. F(2)-isoprostanes, however, were consistently higher in CFS patients.

Identification of a second pseudoautosomal region near the Xq and Yq telomeres.

The telomeres of Xq and Yq have been observed to associate during meiosis, and in rare cases a short synaptonemal complex is present. Molecular cloning of loci from Xqter and Yqter has revealed that their sequence homology extends over 400 kilobases, which suggests the possibility of genetic exchange. This hypothesis was tested by the development of two highly informative microsatellite markers from yeast artificial chromosome clones that carried Xqter sequences and the following of their inheritance in a set of reference pedigrees from the Centre d'Etude du Polymorphisme Humain in Paris, France. From a total of 195 informative male meioses, four recombination events between these loci were observed. In three cases, paternal X alleles were inherited by male offspring, and in one case a female offspring inherited her father's Y allele. These data support the existence of genetic exchange at Xq-Yq, which defines a second pseudoautosomal region between the sex chromosomes.

Family tracing to identify patients with familial hypercholesterolaemia: the second audit of the Department of Health Familial Hypercholesterolaemia Cascade Testing Project.

BACKGROUND: Family tracing is a method recognized to find new patients with familial hypercholesterolaemia (FH). We have implemented family tracing led by FH Nurses and have determined acceptability to patients, feasibility and costs. METHODS: Nurses were located at five National Health Service (NHS) Trusts; they identified FH patients and offered them family tracing. Responses and test results were recorded on a database and summarized on a family pedigree. RESULTS: The majority ( approximately 70%) of index cases participated; the proportion was lower when patients had been discharged from the clinics and in metropolitan areas. On average, 34% (range 13-50%) of relatives lived outside the catchment area of the clinics and could not attend the nurse-led FH clinics. Of the previously untested relatives, 76% who lived in the catchment area of the clinic came forward to be tested. One-third of the relatives who came forward for testing were children

Are patients with familial hypercholesterolaemia well managed in lipid clinics? An audit of eleven clinics from the Department of Health Familial Hypercholesterolaemia Cascade Testing project.

BACKGROUND: Familial hypercholesterolaemia (FH) is an autosomal co-dominant disorder which is relatively common, leads to high levels of LDL-cholesterol and if untreated to early coronary heart disease. An audit of current practice at National Health Service Trusts in England was undertaken to determine whether FH patients meet the diagnostic criteria for FH; are being offered appropriate advice and treatment; and to what extent their families are contacted and offered testing for the disorder. METHODS: Medical records of known FH patients (over 18 years of age and diagnosed before 31 December 2003) were accessed to obtain information on diagnosis, treatment and family tracing. RESULTS: The records of 733 FH patients were examined, 79% met the UK 'Simon Broome' register criteria for the diagnosis of definite or possible FH. Analyses showed that patients were usually offered appropriate advice and treatment, with 89% being on a statin. However, the audit indicated a high variability in family tracing between the sites, with significant differences in the frequency of inclusion of a family pedigree in the notes (range 1-71%, mean 35%); the general practitioner (GP) being advised that first-degree relatives should be tested (range 4-52%, mean 27%); and the proportion of relatives contacted and tested (range 6-50%, mean 32%). CONCLUSION: FH patients are well cared for in lipid clinics in England, are being given appropriate lifestyle advice and medication, but an increase in recording of LDL-cholesterol levels may lead to improvements in their management. Practice in family tracing appears to vary widely between clinics.

Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways.

Heterozygous mutations in NKX2.5, a homeobox transcription factor, were reported to cause secundum atrial septal defects and result in atrioventricular (AV) conduction block during postnatal life. To further characterize the role of NKX2.5 in cardiac morphogenesis, we sought additional mutations in groups of probands with cardiac anomalies and first-degree AV block, idiopathic AV block, or tetralogy of Fallot. We identified 7 novel mutations by sequence analysis of the NKX2.5-coding region in 26 individuals. Associated phenotypes included AV block, which was the primary manifestation of cardiac disease in nearly a quarter of affected individuals, as well as atrial septal defect and ventricular septal defect. Ventricular septal defect was associated with tetralogy of Fallot or double-outlet right ventricle in 3 individuals. Ebstein's anomaly and other tricuspid valve abnormalities were also present. Mutations in human NKX2.5 cause a variety of cardiac anomalies and may account for a clinically significant portion of tetralogy of Fallot and idiopathic AV block. The coinheritance of NKX2.5 mutations with various congenital heart defects suggests that this transcription factor contributes to diverse cardiac developmental pathways.

The human Hsp70B gene at the HSPA7 locus of chromosome 1 is transcribed but non-functional.

The human heat-inducible Hsp70B and Hsp70B' genes were co-localized to 1q23.1 by in situ hybridization. However, though transcripts from Hsp70B could be detected in heat-shocked cells, DNA sequence analyses of both the gene and cDNA copies of the mRNA indicate the gene is non-functional. Moreover, mouse homologues of Hsp70B/B' were not detected by Southern blot analysis, suggesting Hsp70B/B' arose from either Hsp70-1or Hsp70-2 after the divergence of mice and humans.

The DNA sequence of adenovirus type 40.

The 34,214 bp DNA sequence of adenovirus type 40 strain Dugan was determined directly from random fragments of virion DNA cloned into a bacteriophage M13 cloning vector. The gene layout is similar to that of other human adenoviruses, and in addition contains two potential protein-coding regions that are conserved, but have not been recognized previously, in other adenovirus genomes. One is oriented rightward, contained within the intron in the protein-coding region for the L4 33K gene, and would encode a protein sharing N-terminal sequence with 33K. The other is a leftward oriented exon located between the E3 and L5 IV (fibre) regions which would specify the N terminus of a novel protein. The region encoding the C terminus of this protein is not apparent from sequence data.

The DNA sequence of equine herpesvirus 2.

The complete DNA sequence of equine herpesvirus 2 (EHV-2) strain 86/67 was determined. The genome is 184,427 bp in size and has a base composition of 57.5% G + C. Unusually for a herpesvirus, about a third of the sequence distributed in several large blocks appears not to encode proteins. The 79 open reading frames that were identified as probably polypeptide-coding are predicted to encode 77 distinct proteins. Amino acid sequence comparisons confirmed that EHV-2 is a gamma-herpesvirus that is genetically collinear with herpesvirus saimiri (HVS; a gamma 2-herpesvirus) and Epstein-Barr virus (EBV; a gamma 1-herpesvirus), with a closer relationship to the former. Moreover, EHV-2 specifies eight proteins that have counterparts in HVS but not in EBV and only a single protein that has a homologue in EBV but not in HVS (EBV BCRF1, which encodes an interleukin 10-like protein). EHV-2 also encodes three potential G protein-coupled receptors, one with a counterpart in HVS that is specific for alpha chemokines, another with a counterpart in human cytomegalovirus (a beta-herpesvirus), which is specific for beta chemokines, and a third that is assigned more tentatively and lacks detectable counterparts in other herpesviruses.

Improving the referral process for familial breast cancer genetic counselling: findings of three randomised controlled trials of two interventions.

OBJECTIVES: To evaluate the effectiveness and cost-effectiveness of two complementary interventions, using familial breast cancer as a model condition. The primary care intervention consisted of providing computerised referral guidelines and related education to GPs. The nurse counsellor intervention evaluated genetic nurses as substitutes for specialist geneticists in the initial assessment and management of referred patients. DESIGN: The computerised referral guidelines study was a pragmatic, cluster randomised controlled trial (RCT) with general practices randomised to intervention or control groups. The nurse counsellor intervention was tested in two concurrent RCTs conducted in separate UK health service locations, using predetermined definitions of equivalence. SETTING: The computerised referral guidelines trial took place in general practices in Scotland from November 2000 to June 2001. The nurse counsellor intervention took place in a regional genetics clinic in Scotland, and in two health authorities in Wales served by a single genetics service during 2001. PARTICIPANTS: The computerised referral guidelines study involved GPs and referred patients. Both nurse counsellor intervention trials included women referred for the first time, aged 18 years or over and whose main concern was family history of breast cancer. INTERVENTIONS: The software system was developed with GPs, presenting cancer genetic referral guidelines in a checklist approach. Intervention GPs were invited to postgraduate update education sessions, and both intervention and control practices received paper-based guidelines. The intervention period was November 2000 to June 2001. For the nurse counsellor trial, trial 1 ran outpatient sessions with the same appointment length as the standard service offered by geneticists, but the nurse counsellor saw new patients at the first appointment and referred back to the GP or on to a clinical geneticist according to locally developed protocol, under the supervision of a consultant geneticist. The control intervention was the current service, which comprised an initial and a follow-up appointment with a clinical geneticist. In trial 2, a nurse counsellor ran outpatient sessions with the same appointment length as the new consultant-based cancer genetics service and new patients were seen at the first appointment and referred as in trial 1. The control intervention was a new service, and comprised collection of family history by telephone followed by a consultation with a clinical assistant or a specialist registrar, supervised by a consultant. The intervention was implemented between 1998 and 2001. MAIN OUTCOME MEASURES: In the software system trial, the primary outcome was GPs' confidence in their management of patients with concerns about family history of breast cancer. For the nurse counsellor trial, the primary outcome was patient anxiety, measured using standard scales. RESULTS: In the software system trial, 57 practices (230 GPs) were randomised to the intervention group and 29 (116 GPs) to the control group. No statistically significant differences were detected in GPs' confidence or any other outcomes. Fewer than half of the intervention GPs were aware of the software, and only 22 reported using it in practice. The estimated total cost was GBP3.12 per CD-ROM distributed (2001 prices). For the two arms of the nurse counsellor trial, 289 patients (193 intervention, 96 control) and 297 patients (197 intervention and 100 control) consented, were randomised, returned a baseline questionnaire and attended the clinic for trials 1 and 2 respectively. The analysis in both cases suggested equivalence in all anxiety scores, and no statistically significant differences were detected in other outcomes in either trial. A cost-minimisation analysis suggested that the cost per counselling episode was GBP10.23 lower in intervention arm than in the control arm and GBP10.89 higher in the intervention arm than in the control arm (2001 prices) for trials 1 and 2, respectively. Taking the trials together, the costs were sensitive to the grades of doctors and the time spent in consultant supervision of the nurse counsellor, but they were only slightly affected by the grade of nurse counsellor, the selected discount rate and the lifespan of equipment. CONCLUSIONS: Computer-based systems in the primary care intervention cannot be recommended for widespread use without further evaluation and testing in real practice settings. Genetic nurse counsellors may be a cost-effective alternative to assessment by doctors. This trial does not provide definitive evidence that the general policy of employing genetics nurse counsellors is sound, as it was based on only three individuals. Future evaluations of computer-based decision support systems for primary care must first address their efficacy under ideal conditions, identify barriers to the use of such systems in practice, and provide evidence of the impact of the policy of such systems in routine practice. The nurse counsellor trial should be replicated in other settings to provide reassurance of the generalisability of the intervention and other models of nurse-based assessment, such as in outreach clinics, should be developed and evaluated. The design of future evaluations of professional substitution should also address issues such as the effect of different levels of training and experience of nurse counsellors, and learning effects.

Acute lymphoblastic leukemia with an unusual t(8;14)(q11.2;q32): a Pediatric Oncology Group Study.

We present the clinicopathologic findings and survival data on 10 patients with acute lymphoblastic leukemia (ALL) and a rare t(8;14)(q11.2;q32). There were five male and five female patients, nine Caucasians and one Black, aged 4-17 (median 10.9) years. Three had Down syndrome. Eight (80%) patients had a white blood cell (WBC) count <50 x 109/l at presentation. No patient had central nervous system involvement or a mediastinal mass. Two patients had concurrent splenomegaly and hepatomegaly. Adenopathy was absent in four, minimal in three, moderate in one and prominent in two patients. All eight cases where immunophenotyping was performed by flow cytometry showed a B-precursor phenotype with expression of CD10 (CALLA). Only one case exhibited t(8;14)(q11.2;q32) as the sole karyotypic abnormality. Three patients were classified as standard-risk and seven high-risk by NCI (National Cancer Institute) consensus risk group categories. All patients achieved complete remission and seven patients were in complete continuous remission (CCR) after chemotherapy designed for B-precursor ALL. Three patients relapsed after 23.5, 31.3 and 32.1 months of EFS; the first patient also had t(9;22)(q34;q11), the second had a WBC count of 126 x 109/l at presentation while the third patient had no high risk features except for age 10 years. Thus, from our data, the t(8;14)(q11.2;q32) does not appear to confer an increased risk of relapse. Further observations are needed to confirm this conclusion.

Translocations involving chromosome 12p11-13, methotrexate metabolism, and outcome in childhood B-progenitor cell acute lymphoblastic leukemia: a Pediatric Oncology Group study.

Children with B-progenitor cell acute lymphoblastic leukemia whose lymphoblasts at diagnosis accumulate high levels of methotrexate (MTX) and MTX polyglutamates (MTXPGs) appear to have a good prognosis. This has been attributed to increased sensitivity of their blast cells to MTX. However, the proportion of children who are cured of B-progenitor cell acute lymphoblastic leukemia exceeds the number whose lymphoblasts accumulate high MTXPG levels. We report that lymphoblasts from patients with < 50 chromosomes who have translocations that involve the short arm of chromosome 12 accumulate low levels of MTXPGs. These patients appear to have an excellent survival because none of 14 patients with translocations affecting 12p has relapsed, 26-79 months following diagnosis.

Prothrombotic genotypes are not associated with pre-eclampsia and gestational hypertension: results from a large population-based study and systematic review.

DNA samples collected as part of a large population-based case-control study were genotyped to examine the associations of five prothrombotic gene polymorphisms with pre-eclampsia (PE) and gestational hypertension (GH). The polymorphisms studied were: G1691A in Factor V (Factor V Leiden; FVL), prothrombin G20210A, methylenetetrahydrofolate reductase (MTHFR) C677T, plasminogen activator inhibitor-1 4G/5G and the platelet collagen receptor alpha2beta1 C807T. A group of 404 women who developed PE were retrospectively compared with 303 women with GH and 164 control women. The frequency of genotypes did not differ significantly between cases of PE or GH and controls for any of the five polymorphisms studied. We conclude that these prothrombotic genotypes are not associated with the development of PE or GH in our population. The systematic review supports our conclusion, for all but cases of severe disease. which appear to be associated with FVL and, to a lesser extent, MTHFR C677T. There is little value in antenatal screening for prothrombotic polymorphisms to predict the development of pre-eclampsia or gestational hypertension.

Clinical pharmacokinetics of sulindac. A dynamic old drug.

Sulindac is a nonsteroidal anti-inflammatory drug (NSAID) of the indene acetic acid class. The absorption of sulindac is rapid when given orally. Sulindac is reversibly metabolised to sulindac sulphide which has anti-inflammatory and analgesic properties and is irreversibly metabolised to sulindac sulphone which has been suggested to possess antiproliferative effects against tumours. Sulindac and its sulphide and sulphone metabolites bind extensively to plasma albumin. Sulindac is eliminated following bio-transformation; sulindac and sulindac sulphone and their respective glucurooconjugated metabolites are excreted in urine; however only a small amount of the sulindac sulphide metabolite is eliminated in urine. Following long term twice daily administration both sulindac and its metabolites accumulate in plasma. Both patients with cirrhosis and the elderly demonstrate elevated concentrations of all species upon long term sulindac administration as compared with a single dose. The disposition of sulindac and its metabolites may be tied to renal function. In end-stage renal disease, increased free fractions of all species and accumulation of the sulphide and sulphone metabolites, and to a lesser extent sulindac, occurs. Significant drug interactions have been demonstrated for dimethylsulphoxide, cyclosporin, furosemide (frusemide), hydrochlorothiazide, methotrexate and cholestyramine.

Deletion within chromosome 22 is common in patients with absent pulmonary valve syndrome.

Interstitial deletions in chromosome 22 and features associated with CATCH-22 syndrome have been reported in patients with conotruncal congenital heart anomalies. Absent pulmonary valve syndrome is characterized by absent or rudimentary pulmonary valve cusps, absent ductus arteriosus, conoventricular septal defect, and massive dilation of the pulmonary arteries. Because absence of the ductus arteriosus is a key element in the pathogenesis of this syndrome and aortic arch malformations are frequently seen in patients with CATCH-22 syndrome, we hypothesized that patients with absent pulmonary valve syndrome would have a high incidence of deletions in the critical region of chromosome 22. Eight patients with absent pulmonary valve syndrome were studied. Metaphase preparations were examined with fluorescent in situ hybridization of the N25 (D22S75) probe to the critical region of chromosome 22q11.2. Deletions were detected in 6 of 8 patients. The presence of deletions in chromosome 22 in most of the patients we have examined with a diagnosis of absent pulmonary valve syndrome supports a specific genetic and embryologic mechanism involving the interaction of the neural crest and the primitive aortic arches as one cause of congenital absence of the pulmonary valve.

Case of direct insertion within a chromosome 3 leading to a chromosome 3p duplication in an offspring.

An infant with developmental delay and multiple minor congenital anomalies had a duplication of bands p11.1----p14.2 in 3p. Maternal chromosome studies demonstrated a direct intrachromosomal insertion within chromosome 3p.

An 11-year-old boy with mosaic ring chromosome 6 and dilated aortic root.

Autosomal ring chromosomes are rare abnormalities that are inherently unstable. Children with ring chromosome 6 have a wide range of intellectual functioning and congenital anomalies. Cardiac lesions are rarely reported with this chromosome abnormality. We report on a 11-year-old boy with mosaic ring chromosome 6 and a dilated aortic root.

Chromosome abnormalities in congenital heart disease.

Refinements in cytogenetic techniques have promoted progress in understanding the role that chromosome abnormalities play in the cause of congenital heart disease. To determine if mutations at specific loci cause congenital heart disease, irrespective of the presence of other defects, and to estimate the prevalence of chromosome abnormalities in selected conotruncal cardiac defects, we reviewed retrospectively cytogenetic and clinical databases at St. Louis Children's Hospital. Patients with known 7q11.23 deletion (Williams syndrome), Ullrich-Turner syndrome (UTS), and most autosomal trisomies were excluded from this analysis. Two groups of patients were studied. Over a 6.5-year period, 57 patients with chromosomal abnormalities and congenital heart disease were identified. Of these, 37 had 22q11 deletions; 5 had abnormalities of 8p; and 15 had several other chromosome abnormalities. The prevalence of chromosome abnormalities in selected conotruncal or aortic arch defects was estimated by analysis of a subgroup of patients from a recent 22-month period. Chromosome abnormalities were present in 12% of patients with tetralogy of Fallot, 26% in tetralogy of Fallot/pulmonary atresia, 44% in interrupted aortic arch, 12% in truncus arteriosus, 5% in double outlet right ventricle, and 60% in absent pulmonary valve. We conclude that chromosome analysis should be considered in patients with certain cardiac defects. Specifically, fluorescent in situ hybridization (FISH) analysis of 22q11 is indicated in patients with conotruncal defects or interrupted aortic arch. High resolution analysis should include careful evaluation of the 8p region in patients with either conotruncal or endocardial cushion defects.

DNA-based genetic testing in fifty fragile X families.

During the past 4 years (1985-1989), we have analyzed 171 cases in 50 fragile X [fra(X)] families by DNA linkage methods. Most (140 cases; 81%) were for carrier detection, both female (98 cases; 57%) and male (41 cases; 24%). Women who were obligate carriers of the fra(X) mutation accounted for an additional 6 "prior-to-pregnancy" cases. Four pregnancies have subsequently occurred with 3 having been successfully monitored (one male, 2 females). One pregnancy miscarried early prior to testing. Prenatal diagnoses (26 cases; 15%) accounted for the remainder of cases (15 males, 11 females). These will be discussed in the companion paper by Shapiro et al. (Am J Med Genet, 1991). A diagnosis in the cytogenetically uninformative carrier cases was reached in greater than 75% of analyses with a panel of 5 probes: 3 proximal (F9, DXS105, DXS98) and 2 distal (F8, DSX52). Five additional probes, 3 proximal (DXS10, DSX51, DSX102) and 2 distal (DSX15, DXS33), were used in cases that were resistant to analysis with the standard panel. In 60% of cases, flanking markers were identified (proximal and distal). Given this panel, only 5% of cases did not have any informative markers identified. Thus, molecular methods can provide a useful adjunct to cytogenetic analysis in most situations. An unusual association between the rare allele (A1) of DXS10 with the X chromosome carrying the fra(X) mutation was observed. This occurred in both male and female carriers in the uppermost generation tested. The basis for this association is uncertain at the present time.

Congenital heart defect in a patient with deletion of chromosome 7q.

We describe a premature male infant with a terminal deletion of 7q [del(7) (pter----q34:)]. Manifestations include low birth weight, hypertelorism, bilateral cleft lip and palate, cryptorchidism, and a complex congenital heart defect. The latter consisted of hypoplasia of the main pulmonary artery, absent pulmonary valve, ventricular septal defect, and anomalous right pulmonary artery. We briefly review the spectrum of heart defects seen with chromosome 7 deletions, and comment on the incidence of this unusual heart lesion.