De novo variants in CNOT3 cause a variable neurodevelopmental disorder.

As a result of exome-based sequencing work performed by the DDD study, de novo variants in CNOT3 have emerged as a newly recognised cause of a developmental disorder. This paper describes molecular and clinical details of 16 probands with developmental disorders and de novo CNOT3 variants. It is the first such description of the developmental phenotype associated with CNOT3 variants. Eight of these cases were discovered as part of the DDD study, while the other eight were found as a result of large-scale sequencing work performed by other groups. A highly specific phenotype was not recognised in these 16 cases. The most consistent phenotypic features seen in subjects with de novo variants in CNOT3 were hypotonia, relatively small stature, developmental delay, behavioural problems and intellectual disability. There is no easily recognisable facial phenotype, but some common dysmorphic features such as anteverted nares, thin upper lip and low set eyebrows were shared among some of the probands. Haploinsufficiency appears to be the most likely mechanism of action, with eight cases found to have protein-truncating variants. Of the other eight cases (all missense variants), three share an amino acid substitution at the same position which may therefore represent an important functional domain.

Expanding the genotypic spectrum of Perrault syndrome.

Perrault syndrome is a rare autosomal recessive disorder characterized by sensorineural hearing loss (SNHL) in both sexes and primary ovarian insufficiency in 46, XX karyotype females. Biallelic variants in five genes are reported to be causative: HSD17B4, HARS2, LARS2, CLPP and C10orf2. Here we present eight families affected by Perrault syndrome. In five families we identified novel or previously reported variants in HSD17B4, LARS2, CLPP and C10orf2. The proband from each family was whole exome sequenced and variants confirmed by Sanger sequencing. A female was compound heterozygous for a known, p.(Gly16Ser) and novel, p.(Val82Phe) variant in D-bifunctional protein (HSD17B4). A family was homozygous for mitochondrial leucyl aminocyl tRNA synthetase (mtLeuRS) (LARS2) p.(Thr522Asn), previously associated with Perrault syndrome. A further family was compound heterozygous for mtLeuRS, p.(Thr522Asn) and a novel variant, p.(Met117Ile). Affected individuals with LARS2 variants had low frequency SNHL, a feature previously described in Perrault syndrome. A female with significant neurological disability was compound heterozygous for p.(Arg323Gln) and p.(Asn399Ser) variants in Twinkle (C10orf2). A male was homozygous for a novel variant in CLPP, p.(Cys144Arg). In three families there were no putative pathogenic variants in these genes confirming additional disease-causing genes remain unidentified. We have expanded the spectrum of disease-causing variants associated with Perrault syndrome.

Earlier recovery of platelet function after discontinuation of treatment with ticagrelor compared with clopidogrel in patients with high antiplatelet responses.

BACKGROUND: The rate of recovery of platelet function after discontinuation of P2Y(12) inhibitors depends on the reversibility of the antiplatelet effect and the extent of the on-treatment response. P2Y(12) inhibition increases the bleeding risk in patients requiring surgery. OBJECTIVES: To evaluate recovery of platelet function after discontinuation of ticagrelor vs. clopidogrel in stable coronary artery disease (CAD) patients with high levels of platelet inhibition (HPI) during the ONSET/OFFSET study. METHODS: Patients received aspirin 75-100 mg per day and either ticagrelor 90 mg twice-daily or clopidogrel 75 mg daily for 6 weeks. This subanalysis included patients with HPI after the last dose of maintenance therapy, defined as: inhibition of platelet aggregation (IPA) > 75% 4 h post-dose (ADP 20 µm, final extent); < 120 P2Y(12) reaction units 8 h post-dose (VerifyNow P2Y(12) assay); or platelet reactivity index < 50% 8 h post-dose (VASP-P assay). RESULTS: IPA > 75% was observed in 39 out of 47 ticagrelor-treated and 17 out of 44 clopidogrel-treated patients. The rate of offset of IPA over 4-72 h was greater with ticagrelor (IPA %/hour slope: -1.11 vs. -0.67 for clopidogrel; P < 0.0001). Mean IPA was significantly lower with ticagrelor than clopidogrel between 48 and 168 h post-dose (P < 0.01). Similar findings were observed with the other assays. The average time for IPA to decline from 30% to 10% was 50.8 h with ticagrelor vs. 110.4 h with clopidogrel. CONCLUSIONS: In patients with HPI, recovery of platelet function was more rapid after discontinuation of ticagrelor than clopidogrel leading to significantly greater platelet reactivity by 48 h after the last dose in the ticagrelor group.

Barth syndrome: an X-linked cause of fetal cardiomyopathy and stillbirth.

OBJECTIVE: Barth Syndrome (BTHS) is an X-linked multisystem disorder (OMIM 302060) usually diagnosed in infancy and characterized by cardiac problems [dilated cardiomyopathy (DCM) ± endocardial fibroelastosis (EFE) ± left ventricular non-compaction (LVNC)], proximal myopathy, feeding problems, growth retardation, neutropenia, organic aciduria and variable respiratory chain abnormalities. We wished to determine whether BTHS had a significant impact on fetal and perinatal health in a large cohort of family groups originating from a defined region. METHOD: Case note review on 19 families originating from the UK and known to the Barth Syndrome Service of the Bristol Royal Hospital for Children. RESULTS: Details are presented on six kindreds (32%) with genetically and biochemically proven BTHS that demonstrate a wider phenotype including male fetal loss, stillbirth and severe neonatal illness or death. In these families, 9 males were stillborn and 14 died as neonates or infants but there were no losses of females. BTHS was definitively proven in five males with fetal onset of DCM ± hydrops/EFE/LVNC. CONCLUSION: These findings stress the importance of considering BTHS in the differential diagnosis of unexplained male hydrops, DCM, EFE, LVNC or pregnancy loss, as well as in neonates with hypoglycemia, lactic acidosis and idiopathic mitochondrial disease.

CDKL5 mutations cause infantile spasms, early onset seizures, and severe mental retardation in female patients.

OBJECTIVE: To determine the frequency of mutations in CDKL5 in both male and female patients with infantile spasms or early onset epilepsy of unknown cause, and to consider whether the breadth of the reported phenotype would be extended by studying a different patient group. METHODS: Two groups of patients were investigated for CDKL5 mutations. Group 1 comprised 73 patients (57 female, 16 male) referred to Cardiff for CDKL5 analysis, of whom 49 (42 female, 7 male) had epileptic seizure onset in the first six months of life. Group 2 comprised 26 patients (11 female, 15 male) with infantile spasms previously recruited to a clinical trial, the UK Infantile Spasms Study. Where a likely pathogenic mutation was identified, further clinical data were reviewed. RESULTS: Seven likely pathogenic mutations were found among female patients from group 1 with epileptic seizure onset in the first six months of life, accounting for seven of the 42 in this group (17%). No mutations other than the already published mutation were found in female patients from group 2, or in any male patient from either study group. All patients with mutations had early signs of developmental delay and most had made little developmental progress. Further clinical information was available for six patients: autistic features and tactile hypersensitivity were common but only one had suggestive Rett-like features. All had a severe epileptic seizure disorder, all but one of whom had myoclonic jerks. The EEG showed focal or generalised changes and in those with infantile spasms, hypsarrhythmia. Slow frequencies were seen frequently with a frontal or fronto-temporal predominance and high amplitudes. CONCLUSIONS: The spectrum of the epileptic seizure disorder, and associated EEG changes, in those with CDKL5 mutations is broader than previously reported. CDKL5 mutations are a significant cause of infantile spasms and early epileptic seizures in female patients, and of a later intractable seizure disorder, irrespective of whether they have suspected Rett syndrome. Analysis should be considered in these patients in the clinical setting.

Early growth and type 2 diabetes: evidence from the 1946 British birth cohort.

AIMS/HYPOTHESIS: We assessed whether low birthweight or early adiposity rebound was more strongly associated with type 2 diabetes, and whether any effect of low birthweight or early adiposity rebound was explained by adult BMI, adult height, social class of subject or of his/her father, or maternal or paternal diabetes. METHODS: Cox's proportional hazard models were used on data from the National Birth Cohort Study (the MRC National Survey of Health and Development), which was begun in 1946 and had self-reported physician-diagnosed diabetes with age at onset ranging from 31 to 53 years (n=78 cases, and n=47 cases in the multivariate analysis) as the outcome. RESULTS: A U-shaped association between birthweight and type 2 diabetes rates was close to statistical significance (quadratic term p value=0.08). Younger age at adiposity rebound was associated with increased rates of type 2 diabetes (test for trend p=0.002), the association being robust to adjustment for each of sex, birthweight, weight at 2 years, father's social class, parental diabetes, and own social class. The effect of early adiposity rebound was very slightly reduced by adjustment for sex and adult height (p=0.003), but considerably reduced after adjustment for sex and adult BMI (test for trend p=0.1), and further reduced (p=0.4) after additional adjustment for birthweight, weight at 2 years, adult height, social class of subject and of his/her father, and parental diabetes. CONCLUSIONS/INTERPRETATION: Early adiposity rebound was associated with an increased rate of type 2 diabetes independently of birthweight, but its effect was mostly through high adult BMI. Parental diabetes and possibly low weight at 2 years were also risks.

Inherited pericentric inversion (X)(p11.4q11.2) associated with delayed puberty and obesity in two brothers.

We report two brothers with hypogonadotropic hypogonadism (HH), obesity and short stature associated with a maternally inherited pericentric inversion (X)(p11.4q11.2). On the basis that either breakpoint might disrupt a gene whose function is critical to normal sexual development we mapped the chromosomal breakpoints using two-colour fluorescent in situ hybridisation (FISH). The position of both the Xp11.4 and Xq11.2 breakpoints was refined using a panel of ordered BAC clones. No known genes were shown to map to the breakpoint regions. While we cannot entirely exclude the possibility that association between the clinical and cytogenetic phenotypes in the family is coincidental, it is possible that the inversion is responsible for HH through alternative molecular mechanisms such as position effects.

Multilevel investigation of variation in HoNOS ratings by mental health professionals: a naturalistic study of consecutive referrals.

Episodes of mental healthcare in specialist psychiatric services often begin with the assessment of clinical and psychosocial needs of patients by healthcare professionals. Particularly for patients with complex needs or severe problems, ratings of clinical and social functioning at the start of each episode of care may serve as a baseline against which subsequent measures can be compared. Currently, little is known about service variations in such assessments on referrals from primary care. We set out to quantify variability in initial assessments performed by healthcare professionals in three CMHTs in Bristol (UK) using the Health of the Nation Outcome Scales (HoNOS). We tested the hypothesis that variations in HoNOS total and sub-scale scores are related to referral source (general practices), healthcare assessor (in CMHTs) and the assessor's professional group. Statistical analysis was performed using multilevel variance components models with cross-classified random effects. We found that variation due to assessor substantially exceeded that due to referral source (general practices). Furthermore, patient variance differed by assessor profession for the HoNOS--Impairment scores. Assessor variance differed by assessor profession for the HoNOS--Social scores. As HoNOS total and subscale scores show much larger variation by assessor than by referral source, investigations of HoNOS scores must take assessors into account. Services should implement and evaluate interdisciplinary training to improve consistency in use of rating thresholds; such initiatives could be evaluated using these extensions of multilevel models. Future research should aim to integrate routine diagnostic data with continuous outcomes to address selection effects (of patients to assessors) better.

Mutations at the SALL4 locus on chromosome 20 result in a range of clinically overlapping phenotypes, including Okihiro syndrome, Holt-Oram syndrome, acro-renal-ocular syndrome, and patients previously reported to represent thalidomide embryopathy.

We have recently shown that Okihiro syndrome results from mutation in the putative zinc finger transcription factor gene SALL4 on chromosome 20q13.13-13.2. There is considerable overlap of clinical features of Okihiro syndrome with other conditions, most notably Holt-Oram syndrome, a condition in part resulting from mutation of the TBX5 locus, as well as acro-renal-ocular syndrome. We analysed further families/patients with the clinical diagnosis of Holt-Oram syndrome and acro-renal-ocular syndrome for SALL4 mutations. We identified a novel SALL4 mutation in one family where the father was originally thought to have thalidomide embryopathy and had a daughter with a similar phenotype. We also found two novel mutations in two German families originally diagnosed as Holt-Oram syndrome and a further mutation in one out of two families carrying the diagnosis acro-renal-ocular syndrome. Our results show that some cases of "thalidomide embryopathy" might be the result of SALL4 mutations, resulting in an increased risk for similarly affected offspring. Furthermore we confirm the overlap of acro-renal-ocular syndrome with Okihiro syndrome at the molecular level and expand the phenotype of SALL4 mutations.

Siblings with Bohring-Opitz syndrome.

We describe a brother and sister with Bohring-Opitz syndrome and suggest that autosomal recessive inheritance may occur in this condition.

Thrombocytopenia-absent radius syndrome: a clinical genetic study.

The thrombocytopenia-absent radius (TAR) syndrome is a congenital malformation syndrome characterised by bilateral absence of the radii and a thrombocytopenia. The lower limbs, gastrointestinal, cardiovascular, and other systems may also be involved. Shaw and Oliver in 1959 were the first to describe this condition, but it was Hall et al in 1969 who reported the first major series of patients. Since then most reports have been based on single or small numbers of cases. We report the results of a clinical study looking at the phenotype of 34 patients with TAR syndrome. All cases had a documented thrombocytopenia and bilateral radial aplasia, 47% had lower limb anomalies, 47% cow's milk intolerance, 23% renal anomalies, and 15% cardiac anomalies. Congenital anomalies not previously described in association with TAR syndrome included facial capillary haemangiomata, intracranial vascular malformation, sensorineural hearing loss, and scoliosis. Karyotype analysis, chromosome breakage studies including premature centromeric separation and fluorescence in situ hybridisation studies looking for a deletion of chromosome 22q11 were undertaken. Two abnormal karyotypes were identified.

Age period cohort analysis of time trends in regional mortality rates in England, Wales and Scotland.

Previous studies have demonstrated regional variation in mortality rates, this reducing for younger but not older individuals. However, it has not been clear from such studies whether or not the overall findings can or cannot be attributed to particular regions that may have abnormal patterns of change over time. We analyse mortality rates by region using data from the census years from 1931 until 1991 for adults. Age period cohort models, based on the local 'curvatures', are used to describe the changes in the rates. We also use estimable contrasts to compare the later periods with the earlier periods and to compare pre- and post-war cohorts. There is strong evidence that the changes in the mortality rates are associated with non-linear period and cohort effects. There is no evidence that these curvatures vary over regions. There is evidence that the curvatures are not the same among men and women. Among women there is evidence that there is a change in trend in the periods from 1961 to 1991 compared with the trend in the earlier periods from 1931 to 1951, such that the reduction in the mortality rates with time are slowing down for women. Among men who had higher mortality rates, there is not so much evidence of a slowing down of the reduction. Post-war cohorts of women from 1941 onwards are enjoying a reduction in mortality compared with the cohorts from 1901 to 31, while among men there is no evidence of a change in mortality trends among younger cohorts. Changes in the mortality rates over regions have come about from different long-term temporal trends in the regions rather than abrupt changes taking place at different times in different regions. The general convergence of mortality rates over regions over time with a consequent reduction in regional variance is masked by two regions, Scotland and East Anglia. These regions appear to differ from the regions as a whole in the relationship of mortality to age at a given period and in the reduction of mortality over time (drift), Scotland having a higher mortality, especially at younger ages, and lower downward trend, and East Anglia vice versa.

Spectrum, frequency and penetrance of OPA1 mutations in dominant optic atrophy.

Dominant optic atrophy (DOA) is the commonest form of inherited optic neuropathy. Although heterogeneous, a major locus has been mapped to chromosome 3q28 and the gene responsible, OPA1, was recently identified. We therefore screened a panel of 35 DOA patients for mutations in OPA1. This revealed 14 novel mutations and a further three known mutations, which together accounted for 20 of the 35 families (57%) included in this study. This more than doubles the number of OPA1 mutations reported in the literature, bringing the total to 25. These are predominantly null mutations generating truncated proteins, strongly suggesting that the mechanism underlying DOA is haploinsufficiency. The mutations are largely family-specific, although a common 4 bp deletion in exon 27 (eight different families) and missense mutations in exons 8 (two families) and 9 (two families) have been identified. Haplotype analysis of individuals with the exon 27 2708del(TTAG) mutation suggests that this is a mutation hotspot and not an ancient mutation, thus excluding a major founder effect at the OPA1 locus. The mutation screening in this study also identified a number of asymptomatic individuals with OPA1 mutations. A re-calculation of the penetrance of this disorder within two of our families indicates figures as low as 43 and 62% associated with the 2708del(TTAG) mutation. If haploinsufficiency is the mechanism underlying DOA it is unlikely that this figure will be mutation-specific, indicating that the penetrance in DOA is much lower than the 98% reported previously. To investigate whether Leber's hereditary optic neuropathy (LHON) could be caused by mutations in OPA1 we also screened a panel of 28 LHON patients who tested negatively for the three major LHON mutations. No mutations were identified in any LHON patients, indicating that DOA and LHON are genetically distinct.

3-Hydroxyisobutyric aciduria: phenotypic heterogeneity within a single family.

3-Hydroxyisobutyric aciduria is a rare biochemical finding associated with a variable clinical phenotype in the literature. We report two siblings excreting abnormal levels of this metabolite from a consanguineous family who manifested distinct phenotypic variation. We speculate as to whether this biochemical anomaly may simply be an incidental finding and suggest that pre-natal counselling on the basis of metabolite identification may be unwarranted.

p63 Gene mutations in eec syndrome, limb-mammary syndrome, and isolated split hand-split foot malformation suggest a genotype-phenotype correlation.

p63 mutations have been associated with EEC syndrome (ectrodactyly, ectodermal dysplasia, and cleft lip/palate), as well as with nonsyndromic split hand-split foot malformation (SHFM). We performed p63 mutation analysis in a sample of 43 individuals and families affected with EEC syndrome, in 35 individuals affected with SHFM, and in three families with the EEC-like condition limb-mammary syndrome (LMS), which is characterized by ectrodactyly, cleft palate, and mammary-gland abnormalities. The results differed for these three conditions. p63 gene mutations were detected in almost all (40/43) individuals affected with EEC syndrome. Apart from a frameshift mutation in exon 13, all other EEC mutations were missense, predominantly involving codons 204, 227, 279, 280, and 304. In contrast, p63 mutations were detected in only a small proportion (4/35) of patients with isolated SHFM. p63 mutations in SHFM included three novel mutations: a missense mutation (K193E), a nonsense mutation (Q634X), and a mutation in the 3' splice site for exon 5. The fourth SHFM mutation (R280H) in this series was also found in a patient with classical EEC syndrome, suggesting partial overlap between the EEC and SHFM mutational spectra. The original family with LMS (van Bokhoven et al. 1999) had no detectable p63 mutation, although it clearly localizes to the p63 locus in 3q27. In two other small kindreds affected with LMS, frameshift mutations were detected in exons 13 and 14, respectively. The combined data show that p63 is the major gene for EEC syndrome, and that it makes a modest contribution to SHFM. There appears to be a genotype-phenotype correlation, in that there is a specific pattern of missense mutations in EEC syndrome that are not generally found in SHFM or LMS.

The role of comparative genomic hybridisation in prenatal diagnosis.

The aims of this study were to assess the feasibility of using comparative genomic hybridisation instead of conventional cytogenetics in prenatal diagnosis and to determine the size of DNA loss that can be detected. Using comparative genomic hybridisation, six cases with standard aneuploidies were diagnosed correctly. This technique clearly identified a partial duplication of the long arm of chromosome 1 but was not capable of detecting the associated inversion. A small interstitial deletion on short arm of chromosome 10 also was detected precisely. Although the current comparative genomic hybridisation resolution is similar to the sensitivity of the highest resolution G banding, the latter is not a routine strategy in prenatal diagnosis. Comparative genomic hybridisation can allow full chromosome assessment equal to the highest resolution cytogenetic studies without the need for cell culture.

Pancreatic dysfunction in severe obesity.

AIMS: To investigate pancreatic function in children attending an obesity clinic. METHODS: Thirty six children (of which 34 were white) with severe obesity of prepubertal onset (body mass index more than +2 SDS) were reviewed clinically and dysmorphologically, with assessment of pancreatic function. RESULTS: Eight had dysmorphic features and 13 had learning difficulties. Four of 17 prepubertal children had hyperinsulinaemia and seven had hyperproinsulinaemia. All 19 pubertal children had hyperinsulinaemia, 14 had hyperproinsulinaemia, and one had type II diabetes. CONCLUSIONS: Metabolic abnormalities predictive of type II diabetes occur in severely obese white children.

Analysis of falling mortality rates in Edinburgh and Glasgow.

BACKGROUND: The aims of the study were to describe and interpret trends in mortality in Glasgow and Edinburgh METHODS: A comparison was made between observed all-cause and cause-specific mortality rates for 1989-1993 for men and women aged 35-74 and rates predicted on the basis of modelled mortality data for residents of Glasgow and Edinburgh aged 25-74 in quinquennia based on Census years 1961, 1971 and 1981. RESULTS: All-cause mortality rates fell between 1979-1983 and 1989-1993 by a larger amount in Edinburgh than in Glasgow (24.5 versus 14.5 per cent in men; 20.4 versus 10.5 per cent in women). Differences in life expectancy between the cities at age 35 increased by 44 per cent to 4.7 years in men and by 19 per cent to 2.5 years in women. Mortality rates improved in all age and sex groups but trends were least favourable in Edinburgh men and women aged 35-44. Mortality rates in both cities fell by a larger amount than predicted, by 10 per cent in men and 6 per cent in women. CONCLUSIONS: The widening of differences in life expectancy between Glasgow and Edinburgh is mainly due to a historical trend of longevity increasing more quickly in Edinburgh. Although precise explanations are not possible, it seems likely that this difference between the cities is explained in large measure by their consistently and markedly contrasting socio-economic profiles. Comparison of the cities conceals, however, a trend of falling mortality rates in both populations, comprising most of the observed reduction in mortality rates in Glasgow, which appears to result in part from factors operating in the short term. Interpretation of trends in cause-specific mortality rates needs to take account of the possibility of long-term and short-term trends in all-cause mortality in different social groups.

Small area variations in health related behaviours; do these depend on the behaviour itself, its measurement, or on personal characteristics?

In this paper we examine the patterning, by small areas, of four health related behaviours (smoking, alcohol consumption, diet, and exercise) in the West of Scotland, after controlling for a range of individual/household characteristics, using multilevel models. Smoking and drinking were measured both as binary and as continuous variables, and diet and exercise were each measured in two ways: 'good' (health promoting) and 'bad' (health damaging). 'Area effects' (unattributed variation by post code sector) were found for 'bad' diet only. 'Good' and 'bad' diet, 'bad' exercise patterns and current smoking were associated with postcode sector deprivation. For 'bad' diet this effect was found only for individuals in more affluent households, and for 'good' exercise and current smoking the association with area deprivation differed between adolescents and adults. We conclude that the influence of area on health related behaviours varies according to the behaviour and the way it is measured, and that the influence of area deprivation and/or of area can vary by age and household deprivation.