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Hydroacoustic surveys are an important tool for assessing the abundance of Arctic charr (Salvelinus alpinus (L.)) in lakes, the requirement for which is increasing because of new environmental legislation. Consequently, survey demand is now such that assessments must be undertaken with the minimum of effort compatible with scientific validity. One major complication is seasonal variability in abundance, with which surveyors have attempted to deal by defining somewhat arbitrary seasonal sampling windows. The definition of more objective windows was pursued by analysing variations in the seasonal abundance of Arctic charr in Windermere, UK, recorded using day and night hydroacoustic surveys at monthly intervals from 1991 to 2001. Two alternative sampling windows for night surveys free of significant internal seasonal effects were revealed, i.e., December to September and September to December. It was also found that day surveys gave a useful, although lower, estimate of abundance as compared with night surveys.

Summary Background Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the disorder thought most to justify neonatal screening by tandem-mass spectrometry because, without screening, there seems to be substantial morbidity and mortality. Our aim was to assess the overall effectiveness of neonatal screening for MCAD deficiency in Australia. Methods We identified MCAD-deficient patients from a total population of 2 495 000 Australian neonates (810 000 screened) born between April 1, 1994, and March 31, 2004. Those from a cohort of 1 995 000 (460 000 screened) were followed up for at least 4 years, and we recorded number of deaths and severe episodes, medical and neuropsychological outcome, and hospital admissions within the screened and unscreened groups. Findings In cohorts aged at least 4 years there were 35 MCAD-deficient patients in those not screened (2·28 per 100 000 total population) and 24 in the screened population (5·2 per 100 000). We estimated that patients with this disorder in the unscreened cohort remained undiagnosed. Before 4 years of age, three screened patients had an episode of severe decompensation (including one neonatal death) versus 23 unscreened patients (including five deaths). At the most conservative estimate, relative risk of an adverse event was 0·44 (95% CI 0·13–1·45). In the larger cohort the relative risk (screened vs unscreened) of an adverse event by age 2 years was 0·26 (95% CI 0·07–0·97), also a conservative estimate. 38 of 52 living patients had neuropsychological testing, with no suggestions of significant differences in general cognitive outcome between the groups. Interpretation Screening is effective in patients with MCAD deficiency since early diagnosis reduces deaths and severe adverse events in children up to the age of 4 years.

We present two siblings with a previously undescribed congenital disorder of glycosylation (CDG). The first child died in utero with severe hydrops fetalis and the second presented following preterm delivery with respiratory insufficiency, generalised edema and a protein-losing enteropathy. Both had a similar pattern of facial dysmorphism and joint contractures. The diagnosis of CDG-I was made following the birth of the second child based on the serum transferrin isoform pattern. CDG-Ia and -Ib were excluded by specific enzyme analysis. Joint contractures are a relatively uncommon finding in CDG, although fetal hydrops (CDG-Ia) and protein-losing enteropathy (CDG-Ib) are well recognized. CDG must be considered in the differential diagnosis of hydrops fetalis, congenital hypoproteinemia and death in early infancy, particularly when associated with dysmorphic features.

Idiopathic hyperammonemic encephalopathy is an uncommon but frequently fatal complication of chemotherapy. It is characterised by abrupt alteration in mental status with markedly elevated plasma ammonia levels in the absence of obvious liver disease or any other identifiable cause, and frequently results in intractable coma and death. It usually occurs in patients with haematologic malignancies during the period of neutropenia following cytoreductive therapy or bone marrow transplantation, and in solid organ malignancies treated with 5-fluorouracil. Although the aetiology of this syndrome is yet to be determined, it appears to be multi-factorial in nature. Optimal management remains to be formally established, and the critical step is increased awareness of the syndrome by measurement of plasma ammonium levels in patients with neurological symptoms, leading to early diagnosis and the prompt implementation of therapy.

Background Glucosylsphingosine (GluSph) has emerged as a biomarker for the inherited metabolic disorder, Gaucher disease (GD). We developed a simple laboratory test to measure plasma GluSph and show that elevated GluSph is diagnostic for GD as well as informing on disease burden for monitoring patients on treatment. Methods GluSph was measured from a single-phase total lipid extraction of 0.01 mL of plasma by liquid chromatography–electrospray ionisation–tandem mass spectrometry and concentrations extrapolated from a seven point standard curve (0.04 to 20 pmoL). A total of 1464 samples were tested and longitudinal assessment of an additional 20 GD patients. Results All patients with GD had elevated GluSph compared to unaffected controls and 16 other metabolic disorders. GluSph was also slightly elevated in three patients with Krabbe disease but not at concentrations to confuse a GD diagnosis. GluSph correlated with chitotriosidase in the majority of GD patients on treatment who were informative for this marker. Conclusions GluSph can be easily measured from 0.01 mL of plasma and is useful as a diagnostic marker for GD with the current platform suited to high-throughput screening. It outperforms other GD biomarkers for biochemical monitoring of patients receiving enzyme replacement therapy for all individuals.

Introduction In this study, we aimed to evaluate whether fasting plasma citrulline concentration predicts subsequent glucose absorption in critically ill patients. Methods In a prospective observational study involving 15 healthy and 20 critically ill subjects, fasting plasma citrulline concentrations were assayed in blood samples immediately prior to the administration of a liquid test meal (1 kcal/ml; containing 3 g of 3-O-methylglucose (3-OMG)) that was infused directly into the small intestine. Serum 3-OMG concentrations were measured over the following 4 hours, with the area under the 3-OMG concentration curve (AUC) calculated as an index of glucose absorption. Results The groups were well matched in terms of age, sex and body mass index (BMI) (healthy subjects versus patients, mean (range) values: age, 47 (18 to 88) versus 49 (21 to 77) years; sex ratio, 60% versus 80% male; BMI, 25.2 (18.8 to 30.0) versus 25.5 (19.4 to 32.2) kg/m2). Compared to the healthy subjects, patients who were critically ill had reduced fasting citrulline concentration (26.5 (13.9 to 43.0) versus 15.2 (5.7 to 28.6) μmol/L; P

Spawning is a key but often fragile event in the life cycles of fish populations. Nevertheless, it has been relatively little studied for lithophilic lacustrine species requiring hard spawning substrates, such as gravels or stones, largely devoid of fine sediments. Twelve demonstrated or putative spawning grounds of Arctic charr (Salvelinus alpinus) in shallow and deep areas of the north and south basins of the eutrophicated lake of Windermere, UK, were described by hydroacoustic, physical and visual surveys. In addition, their current conditions were compared with their original qualitative descriptions made over 50 years ago. Spawning ground characteristics were found to be more complex than originally described, with considerable overlaps in depth ranges and only limited areas of appropriate hard substrates. Moreover, extensive gill netting surveys in recent years have found spawning Arctic charr at only seven of the original 12 demonstrated or putative spawning grounds, although several new spawning areas have also been found. The distribution of unsuitable fine sediments is widespread in the lake, particularly in the more eutrophicated south basin where suitable spawning habitat within the putative spawning areas is limited. Windermere faces a number of environmental problems including climate change and species introductions. However, the temporal and spatial patterns of the lake's eutrophication suggest that associated increases in fine sediments have been a major driver of the observed deterioration of Arctic charr spawning grounds and so may have also contributed to a marked decline recently observed in the local abundance of this species.

Reduction of an enzyme activity required for the lysosomal degradation of glycosaminoglycan (gag) chains will result in a mucopolysaccharidosis (MPS) disorder. Substrate deprivation therapy (SDT), a potential therapy option for MPS with residual enzyme activity, aims to reduce the synthesis of gag chains, the natural substrate for the deficient enzyme. Reduced substrate levels would balance the reduced level of enzyme in patient cells, resulting in normalized gag turnover. Rhodamine B, a nonspecific inhibitor, reduced gag synthesis in a range of normal and MPS cells and also decreased lysosomal storage of gag in MPS VI (72%) and MPS IIIA (60%) cells. Body weight gain of male MPS IIIA mice treated with 1 mg/kg rhodamine B was reduced compared with untreated MPS IIIA mice and was indistinguishable from that of normal mice. Liver size, total gag content, and lysosomal gag was reduced in treated MPS IIIA animals as was urinary gag excretion. Lysosomal gag content in the brain was also reduced by treatment. The alteration in MPS IIIA clinical pathology by rhodamine B, combined with the observation that treatment had no effect on the health of normal animals, demonstrates the potential for SDT in general as a therapy for MPS disorders.

Blood was obtained by cordocentesis from a fetus with non-immune hydrops demonstrated by ultrasound scanning at 27 weeks' gestation. Abnormalities of serum transferrin isoelectric focussing (IEF) were identified, characteristic of a congenital disorder of glycosylation type I (CDG-Ia). A diagnosis of CDG-Ia was confirmed by enzyme analysis of cultured amniocytes. This is the first report of CDG-Ia diagnosed by serum analysis in a fetus. Previous reports have warned that diagnostic abnormalities do not appear in serum until several weeks after birth. The sensitivity of cordocentesis transferrin IEF is unknown but is less than 100% effective because cases have been diagnosed postnatally after normal prenatal or neonatal studies. Enzyme analysis or mutation analysis is required for diagnosis of congenital disorder of glycosylation (CDGs) regardless of whether a diagnostic transferrin pattern is identified prenatally. The analysis of a small sample of serum, from cordocentesis, performed to check for fetal anemia, simplified the investigation, diagnosis, and genetic counselling of a case of non-immune hydrops detected at 27 weeks' gestation. This might be a useful test for other cases in these circumstances, as fetal blood is usually collected to check for anemia.

Long-term data from the perch population of the north basin of Windermere, UK, were combined with effects data from laboratory toxicity studies of survival, growth and reproduction in other species to assess the likely impact on fish populations of different levels of exposure to nonylphenol (NP) and ethinylestradiol (EE2). A multi-stage Delay Differential Equation fish population model was used to simulate the perch population during two periods when it showed contrasting population structures (1968–1973 and 1983–1988) as the result of a disease outbreak in 1976, and to extrapolate the effects of chemical exposure to EE2 and NP observed in the laboratory to the environment. In the absence of chemical exposure, model simulations predicted population numbers (females) in line with those derived from field survey data. Effects predictions were made for long-term exposure (20 years) to low and high doses of EE2 (1 and 10 ng l−1) and NP (1 and 30 μg l−1). The sustained high-level exposure of EE2 had a high probability of causing the extinction of a confined fish population such as that in Windermere, however, such an exposure scenario is unlikely. Far greater uncertainty surrounds the prediction of effects due to low-level exposure of fish populations and even though effects may appear to be significant in laboratory studies, such as fecundity lowered by 30%, they may not necessarily translate into significant population effects in the field. This is especially true if life-history data show high natural variability in terms of individual vital rates. Our work suggests that for a decline in the perch population numbers to be significant in Windermere, it would have to be substantially more than 50%. Our model predictions indicated that the post-disease population was generally more vulnerable than the pre-disease population and had a significantly greater probability of declining by 50% following single chemical exposure, but both populations were equally likely to decline following multiple chemical exposure. Rate of population recovery was shown to be a more sensitive measure in terms of differentiating the effects of low and high chemical exposure as well as the vulnerability of populations with contrasting structures, histories or levels of background stress.

Aim: To study the effectiveness of a specific national programme of enzyme replacement therapy (ERT) for patients with severe forms of Gaucher disease, a disorder of sphingolipid metabolism resulting from an inherited deficiency of the lysosomal enzyme β-Glucocerebrosidase. Methods: Prospective analysis of data submitted at entry and every 6 months on therapy. The responses of haemoglobin (Hb) and platelet (plt) concentrations, liver and spleen volumes were assessed. Patients: Forty-eight patients were treated with ERT for a minimum of 6 months. Forty patients had Type 1 disease and eight had Type 3B. The age range was 1–70 years (median 24 years). Duration of therapy at the time of analysis was 6–114 months. Results: Thirty-six per cent of patients started with a normal Hb increasing to 76% after 6 months. The mean improvement in Hb from baseline to the end of study period was 20 g/L, when the Hb was normal in 85% (41 patients). Thirty per cent of patients had a normal plt count at the start of therapy, with a more gradual increase in the count at 6 monthly intervals of 50, 91, 108 and 142% of starting value. Seventy-five per cent of patients had a normal plt count at the end of study. Spleen volumes reduced by a mean of 56% in 33 evaluable patients, and the liver by 27% in 30 of 38 evaluable patients. Eight patients had an increase in liver volume of 28%. Conclusion: Enzyme replacement therapy produced a spectrum of beneficial responses in patients with Gaucher disease, but all had some evidence of reversal of haematological complications and/or reduction in visceromegaly. Future analyses will examine the effect of therapy on bone disease, prepubertal growth and quality of life. (Intern Med J 2005; 35: 156–161)

A notable deficiency in the use of tandem mass spectrometry (MS/MS) for newborn screening is the inability to screen for urea cycle defects. The most common of these, with an incidence of 1 in 14 000 births (1), is the inherited X-linked disorder ornithine transcarbamylase deficiency (OTCD). A majority (60%) of hemizygous males risk death from hyperammonemic coma during the first week of life. The remainder, including 10% of heterozygous females, exhibit lethargy, vomiting episodes, and behavioral problems during childhood. The severity of the disorder and the potential for correction of OTCD by liver transplantation and gene therapy (2) provide adequate justification for newborn screening. OTCD patients have low blood citrulline because of reduced conversion from carbamoyl phosphate. Citrulline is one of the amino acids routinely measured in MS/MS newborn-screening programs. Unfortunately, many protein-restricted newborns also have low blood citrulline (3). A more selective amino acid metabolite for OTCD is glutamine. The derivatization procedure used in many MS/MS screening programs (4), which uses butanol–hydrogen chloride, destroys glutamine. Approximately one-half of the glutamine is converted to glutamic acid dibutyl ester and is indistinguishable from that formed from endogenous glutamic acid in the blood. The surviving glutamine butyl ester is deaminated in acidic solution to a protonated form of pyroglutamic acid butyl ester in the electrospray source of the MS/MS. Again it is not possible to distinguish this pyroglutamic acid from what is already present in the blood. As a secondary consequence, the measurements of glutamic and pyroglutamic (and by analogy, aspartic) acids in blood spots after derivatization are grossly inaccurate. MS/MS newborn-screening programs that do not derivatize amino acids avoid solvolysis of glutamine and of pyroglutamic acid to glutamic acid. During electrospray ionization-MS/MS analysis, however, glutamine is again indistinguishable from pyroglutamic acid. Resolution is possible by separation with time-consuming liquid chromatography …

Objectives: To determine the proportion of urine amino and organic acids screening tests (UMS) undertaken for patients referred with developmental delay or intellectual disability (DD/ID), and within the group with DD/ID, to determine the diagnostic yield, the proportion of diagnoses with a therapy and the associated recurrence risks. Methods: A retrospective review of request forms and results of UMS, in individuals older than 28 days, referred to the Women's and Children's Hospital, North Adelaide, between 1 January 1992 and 31 December 1998 was carried out. Urine was analysed by ion exchange chromatography (amino acids), gas chromatography/mass spectrometry (organic acids), colorimetric assay (orotic acid) and stable isotope−dilution mass spectrometry (trimethylamine). Results: A total of 3316 samples were received, 1447 being from patients with DD/ID. A diagnosis was determined for 1.8% of all referrals. For patients with DD/ID, the diagnostic yield was 1.1%, with a similar yield for isolated DD/ID and DD/ID with other features (9/828 vs 7/619; χ2 = 0.006; P = 0.93). Specific therapies were available for 69% of diagnoses associated with DD/ID and 87.5% had known Mendelian or mitochondrial inheritance. Conclusion: Urine metabolic screening is an important part of the evaluation of children with DD/ID as it can enable families to make reproductive decisions and children to receive appropriate therapy early.

Cystathionine beta-synthase (CBS) deficiency is the most common cause of homocystinuria. It is inherited as an autosomal recessive trait and common clinical features are: dislocation of the optic lens, osteoporosis, mental retardation, and thromboembolism. We determined the molecular basis of CBS deficiency in 36 Australian patients from 28 unrelated families, using direct sequencing of the entire coding region of the CBS gene. The G307S and I278T mutations were the most common mutations. They were present in 19% and 18% of independent alleles, respectively. In total, seven novel and 20 known mutations were detected. Of those, the two novel missense mutations (C109R and G347S), as well as two known missense mutations (L101P and N228K), were expressed in E. Coli. All mutant proteins completely lacked catalytic activity. Furthermore, we studied the correlation between genotype and the biochemical response to pyridoxine treatment in the patients of whom 13 were pyridoxine responsive, 21 were non-responsive, and two were partially responsive. The G307S mutation always resulted in a severe non-responsive phenotype, whereas I278T resulted in a milder B6 responsive phenotype. From our results, we were also able to establish three other mild mutations: P49L, R369C, and V371M.

Methylmalonic aciduria is a rare disorder of organic acid metabolism with limited therapeutic options, resulting in high morbidity and mortality. Positive results from combined liver/kidney transplantation suggest, however, that metabolic sink therapy may be efficacious. Gene therapy offers a more accessible approach for the treatment of methylmalonic aciduria than organ transplantation. Accordingly, we have evaluated a lentiviral vector-mediated gene transfer approach in an in vivo mouse model of methylmalonic aciduria. A mouse model of methylmalonic aciduria (Mut(-/-)MUT(h2)) was injected intravenously at 8 weeks of age with a lentiviral vector that expressed a codon-optimized human methylmalonyl coenzyme A mutase transgene, HIV-1SDmEF1αmurSigHutMCM. Untreated Mut(-/-)MUT(h2) and normal mice were used as controls. HIV-1SDmEF1αmurSigHutMCM-treated mice achieved near-normal weight for age, and Western blot analysis demonstrated significant methylmalonyl coenzyme A enzyme expression in their livers. Normalization of liver methylmalonyl coenzyme A enzyme activity in the treated group was associated with a reduction in plasma and urine methylmalonic acid levels, and a reduction in the hepatic methylmalonic acid concentration. Administration of the HIV-1SDmEF1αmurSigHutMCM vector provided significant, although incomplete, biochemical correction of methylmalonic aciduria in a mouse model, suggesting that gene therapy is a potential treatment for this disorder.

Neonatal screening is an essential preventative public health program, and is the standard practice of care world– wide. India is yet to start any publicly funded program despite this having been established in many countries for over 50 years. The purpose of neonatal screening is to reduce morbidity and mortality in the newborn. Detection must have a clear benefit for the baby and be cost effective when compared to the cost associated with delayed treatment. These criteria are clearly satisfied for screening a baby at birth for congenital hypothyroidism (CHT). Though systematic neonatal screening for congenital hypothyroidism was introduced in the early 1970 in many countries, in India an estimated 10,000 babies are born with congenital hypothyroidism every year, yet there is no screening program for this. We present the details of our study, which reveals that CHT in India has a high incidence and is an URGENT high priority for public screening.

We report a patient with late-onset nonketotic hyperglycinaemia managed with a sequential approach to drug therapy in placebo-controlled therapeutic trials. Partial response to low-protein diet and sodium benzoate and dramatic response to imipramine are demonstrated, with parental scores on the Developmental Behavioural Checklist falling from the 86th centile before treatment to normal with combined benzoate and imipramine therapy.

– Analysis of the Windermere (England), Perca fluviatilis (1965–1997) and Esox lucius (1943-1997) fishery data indicates there have been substantial changes in sex ratios through time. In the case of perch, a 1976 perch disease outbreak substantially skewed the sex ratio in favor of females. It took over a decade for the fishery to recover to its pre-1976 state. In the case of pike, both catch and year-class data are female biased, but this bias has significantly weakened through time. This trend towards proportionately more males through time may reflect an effect of increasing spring temperatures or an evolutionary or phenotypic response to female-biased fishing pressure.

Our objective was to review the Australasian experience of X-linked adrenoleukodystrophy (ALD), to compare the spectrum of disease seen in Australasia with previously published data from elsewhere, and to assess the reliability of carrier testing. Study design was a retrospective review of records collected over a 15-year period, the setting was an international referral laboratory for the study of metabolic disease, and the subjects were all known cases of ALD diagnosed in Australia and New Zealand between 1981 and 1996 and their families. We estimate that the combined incidence of ALD and its variants in Australasia is at least 1.6 per 100,000. Of 95 affected males, 51 had cerebral adrenoleukodystrophy, 24 had adrenomyeloneuropathy, 15 had Addison's disease only, and 5 remained asymptomatic when last examined. However, the distribution of phenotypes among newly diagnosed patients has changed substantially over the last 15 years, with cerebral forms of the disease forming a decreasing proportion of new diagnoses. The measurement of plasma very long chain fatty acids (VLCFAs) alone detects 93% of women who can be proven to be carriers. The addition of genetic linkage studies or assay of VLCFAs in cultured fibroblasts improved this detection rate to the point that there were no obligate carriers who could not be detected using a combination of two or more techniques.

From July 1989 to December 1994, an echo sounder provided monthly estimates, usually for both day and night, of pelagic salmonid densities in the North and South Basins of Windermere, the largest natural lake in England. Sampling was along contiguous transects, three in the North Basin and five in the South Basin. Records for Arctic charr (Salvelinus alpinus) could not be separated from those for brown trout (Salmo trutta), but previous sampling by gill-nets and anglers showed that charr formed over 90% of this mixed population in the North Basin and about 60–75% in the South Basin. Associated with the increasing eutrophication of the lake, there has been a decline in anglers' catches of charr and, since 1984, an increase in brown trout taken in the pelagic zone of the South Basin. The echo-sounder data showed that pelagic salmonid density in the North Basin was about two to five times that in the more eutrophic South Basin in 1989, 1990 and 1991. Since the start, in April 1992, of the reduction of phosphorus discharged from sewage works, this ratio has decreased, especially at night when the highest densities were recorded. This improvement was chiefly due to a significant (P 20 m) water layers. Although a similar improvement has still to be shown in the upper water layer by larger fish above the size limit for removal by angling (20 cm), there has been a significant increase (P 20 cm at water depths

Prostaglandin (PG)D2has been shown to be an active agent in the resolution of experimentally induced inflammation. This study was undertaken to determine the presence of PGD2in chronic joint effusions and to explore the potential contributions of dendritic cells (DC) and monocytes to the intra-articular synthesis of PGD2. Synovial fluid (SF) was obtained from patients with inflammatory arthritis and knee effusions. PGD2and PGE2were detected in SF by ultrahigh-performance tandem mass spectrometry. Cellular fractions in SF were separated by density-gradient centrifugation and flow cytometry. The expression of hematopoietic prostaglandin D-synthase (hPGDS) and PGE-synthase (PGES) mRNA was determined by RT-PCR. Both PGD2and PGE2were detected in blood and SF, with PGD2being more abundant than PGE2in SF. mRNA for hPGDS was more abundant in SF mDCs than SF monocytes (P<0.01) or PB monocytes (P<0.001). SF mDC expressed significantly more hPGDS than PGES. Expressions of PGD2and hPGDS were inversely associated with serum C-reactive protein (P<0.01) and erythrocyte sedimentation rate (P<0.01). The findings suggest that synovial DCs may be an important source of hPGDS and that systemic disease activity may be influenced by actions of PGD2in RA and other arthropathies.

The interruption of the manufacturing process of agalsidase beta has led to a worldwide shortage of this drug. In the EU, nearly all patients initially reduced their agalsidase beta dose, and many of these switched to agalsidase alfa (Replagal Shire HGT). The clinical consequences of this period of drug shortage need to be further evaluated. A gradual increase of agalsidase beta supply is now expected. This implies that patients could resume or even commence agalsidase beta treatment. Guidance for prioritization of patients is needed to support equitable distribution of agalsidase beta to EU member states. To achieve this, in absence of level I clinical evidence, a draft consensus proposal was initiated and distributed. No full consensus was achieved, as there is disagreement regarding the indications for switching patients from agalsidase alfa to agalsidase beta. Some physicians support the concept that the 1.0 mg/kg EOW dose of agalsidase beta is more effective than agalsidase alfa at 0.2 mg/kg EOW, while others believe that at recommended dose, the preparations are equivalent. In light of these difficulties and the uncertainties with respect to supply of agalsidase beta, recommendations were agreed upon by a subgroup of physicians. These current recommendations focus on prioritization of criteria indicative of disease progression.

We use six decades of catch-at-age data for perch (Perca fluviatilis) and pike (Esox lucius) in Windermere (UK) to estimate age- and sex-specific population sizes, natural mortalities and catchabilities in both species. Population sizes are estimated by fitting age structured population models to the catch-at-age data using standard maximum likelihood methods. We validate our methods using data simulations, and use our estimates of vital rates (natural mortality, recruitment and catahability) to address important aspects of fisheries biology. Our model indicates that strong fishery selection against male perch apparently triggered a population collapse, highlighting that sex-selective fisheries can be harmful even at a reasonable exploitation rate (here ≤30%). Recruitment (R) increased with the abundance of spawners (S) in both species, but it also responded to both abiotic and other biotic factors. In particular, increased predator (pike) abundance induced a change from compensation to depensation in the prey (perch) SR relationship, thus favouring the occurrence of an Allee effect. Our study provides reference points for the effective exploitation of pike and perch populations, and underscores the need for ecosystem-based harvesting management.

Anthropogenic factors, including climate warming, are increasing the incidence and prevalence of infectious diseases worldwide. Infectious diseases caused by pathogenic parasites can have severe impacts on host survival, thereby altering the selection regime and inducing evolutionary responses in their hosts. Knowledge about such evolutionary consequences in natural populations is critical to mitigate potential ecological and economic effects. However, studies on pathogen-induced trait changes are scarce and the pace of evolutionary change is largely unknown, particularly in vertebrates. Here, we use a time series from long-term monitoring of perch to estimate temporal trends in the maturation schedule before and after a severe pathogen outbreak. We show that the disease induced a phenotypic change from a previously increasing to a decreasing size at maturation, the most important life-history transition in animals. Evolutionary rates imposed by the pathogen were high and comparable to those reported for populations exposed to intense human harvesting. Pathogens thus represent highly potent drivers of adaptive phenotypic evolution in vertebrates.

Genomic testing using gene panels and whole-exome sequence data are becoming increasingly attractive options to support the clinical management of patients. Whole-exome sequencing to diagnose rare genetic disorders in paediatrics offers a cost-effective and efficient first screen for cases where there is no obvious clinical diagnosis, thus fundamentally shifting medical service provision paradigms. In other areas of medicine, the implementation of targeted gene panels using next generation sequencing is associated with improved diagnosis rate and faster time to diagnosis. Successful integration of genomic testing into the patient management pathway requires updating of the traditional models of test provision. Important factors to be considered include: who should qualify for what genomic test, payment for testing, the appropriate referral mechanism, and how to effectively communicate the results and limitations of genomic tests to patients and clinicians. We discuss strategies and give examples from implementing gene panels and whole-exome sequencing in clinical diagnostics to support the management of patients in South Australia. The next few years are likely to be an exciting and challenging time in genomic test provision in Australia, as these new platforms establish themselves as part of routine medical care and different stakeholders (local, international, direct-to-consumer) define their share of the clinical sequencing market.

Estimated pike Esox lucius recruitment varied by a factor of 16 for females from 1944 to 1991 and by a factor of 27 for males from 1943 to 1990 in Windermere, a temperate, mesotrophic U.K. lake. No significant stock–recruitment relationships were found, but analysis with general additive models (GAMs) revealed that early autumnal water temperature, strength and direction of the North Atlantic Oscillation displacement (corresponding to different climatic conditions in winter) and zooplankton abundance but above all, late summer water temperature were important explanatory variables over the entire time series. Female recruitment was also influenced by young-of-the-year winter temperature. There was no evidence that perch Perca fluviatilis year-class strength, lake level or the summer position of the Gulf Stream influenced recruitment. The fitted models explained up to c. 65% of the overall observed variation between years.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder caused by a deficiency in sulphamidase (NS), a lysosomal enzyme required for the degradation of heparan sulphate glycosaminoglycans (gags). The MPS IIIA mouse is a naturally occurring model that accurately reflects the human pathology and disease course. It displays primarily central nervous system pathology accompanied by widespread accumulation of gag in somatic tissues. MPS IIIA mice exhibit greater bodyweight gain than normal littermates and attain a higher mature bodyweight. In this study, gastrointestinal morphology and function was characterised in the IIIA mouse. Stomach and duodenum weight increased in MPS IIIA mice and duodenum length also increased. An increased submucosal thickness was observed in MPS IIIA intestine compared to normal mice and lysosomal storage of gag was observed in this region. Storage was also observed in the lamina propria of the villus tip. All other morphometric measurements including villus height and crypt depth fell within the normal range. The gastric emptying half-life of solid and liquid meals decreased with age in normal mice whereas the T½ of solid meals did not alter with age in MPS IIA mice such that they were elevated above normal by 38 weeks of age. Sucrase activity was higher than normal in MPS IIIA at all ages tested. These abnormalities in GI structure and function observed in MPS IIIA may contribute to weight gain in this disorder. Refereed/Peer-reviewed

Selection can alter predator-prey interactions. However, whether and how complex food-webs respond to selection remain largely unknown. We show in the field that antagonistic selection from predators and pathogens on prey body-size can be a primary driver of food-web functioning. In Windermere, U.K., pike ( Esox lucius , the predator) selected against small perch ( Perca fluviatilis , the prey), while a perch-specific pathogen selected against large perch. The strongest selective force drove perch trait change and ultimately determined the structure of trophic interactions. Before 1976, the strength of pike-induced selection overrode the strength of pathogen-induced selection and drove a change to larger, faster growing perch. Predation-driven increase in the proportion of large, infection-vulnerable perch presumably favored the pathogen since a peak in the predation pressure in 1976 coincided with pathogen expansion and a massive perch kill. After 1976, the strength of pathogen-induced selection overrode the strength of predator-induced selection and drove a rapid change to smaller, slower growing perch. These changes made perch easier prey for pike and weaker competitors against juvenile pike, ultimately increasing juvenile pike survival and total pike numbers. Therefore, although predators and pathogens exploited the same prey in Windermere, they did not operate competitively but synergistically by driving rapid prey trait change in opposite directions. Our study empirically demonstrates that a consideration of the relative strengths and directions of multiple selective pressures is needed to fully understand community functioning in nature.

Selective harvest of large individuals should alter natural adaptive landscapes and drive evolution toward reduced somatic growth and increased reproductive investment. However, few studies have simultaneously considered the relative importance of artificial and natural selection in driving trait changes in wild populations. Using 50 years of individual-based data on Windermere pike ( Esox lucius ), we show that trait changes tracked the adaptive peak, which moved in the direction imposed by the dominating selective force. Individual lifetime somatic growth decreased at the start of the time series because harvest selection was strong and natural selection was too weak to override the strength of harvest selection. However, natural selection favoring fast somatic growth strengthened across the time series in parallel with the increase in pike abundance and, presumably, cannibalism. Harvest selection was overridden by natural selection when the fishing effort dwindled, triggering a rapid increase in pike somatic growth. The two selective forces appear to have acted in concert during only one short period of prey collapse that favored slow-growing pike. Moreover, increased somatic growth occurred concurrently with a reduction in reproductive investment in young and small female pike, indicating a tradeoff between growth and reproduction. The age-specific amplitude of this change paralleled the age-specific strength of harvest pressure, suggesting that reduced investment was also a response to increased life expectancy. This is the first study to demonstrate that a consideration of both natural selection and artificial selection is needed to fully explain time-varying trait dynamics in harvested populations.

The ability of natural selection to drive local adaptation has been appreciated ever since Darwin. Whether human impacts can impede the adaptive process has received less attention. We tested this hypothesis by quantifying natural selection and harvest selection acting on a freshwater fish (pike) over four decades. Across the time series, directional natural selection tended to favour large individuals whereas the fishery targeted large individuals. Moreover, non-linear natural selection tended to favour intermediate sized fish whereas the fishery targeted intermediate sized fish because the smallest and largest individuals were often not captured. Thus, our results unequivocally demonstrate that natural selection and fishery selection often acted in opposite directions within this natural system. Moreover, the two selective factors combined to produce reduced fitness overall and stronger stabilizing selection relative to natural selection acting alone. The long-term ramifications of such human-induced modifications to adaptive landscapes are currently unknown and certainly warrant further investigation.

For over two decades gene therapy has been actively pursued as a treatment modality for the inherited diseases that affect the paediatric population, however, it is still to make a real impact in the clinic. There are many reasons for this including inadequate technology and a lack of understanding of the biological complexities that impact on the efficiency of gene delivery and its outcomes, both positive and negative. However, recent progress is now addressing these issues and indicates that these problems can be overcome, and that gene therapy will play a significant role in the treatment of at least some of these disorders. This review will first give a short overview of relevant gene delivery technologies, what strategies can be used and which diseases are potential targets for gene therapy, and then illustrate several specific diseases for which gene therapy is actively being developed.

Mucopolysaccharidosis type IIIA (MPS IIIA) is a specific lysosomal storage disorder caused by an enzyme deficiency in sulphamidase, which is required for the degradation of heparan sulphate glycosaminoglycan (gag). This deficiency results in widespread gag storage and leads to severe CNS degeneration and mild somatic pathology. We have developed substrate deprivation as a therapy (SDT) for MPS disorders to reduce the initial production of gag substrate for the deficient enzyme, using the compound rhodamine B as an inhibitor of gag biosynthesis. This should restore the balance between gag level and residual enzyme activity towards normal and improve patient outcome. To determine if SDT improved CNS function, MPS IIIA mice were treated for 6 months with weekly, intravenous 1 mg/kg rhodamine B and then tested in a 4-arm water cross maze, which measures spatial learning and memory. MPS IIIA untreated mice were unable to perform to the same level as normal littermates, having increased escape latency, increased incorrect entries and decreased correct entries. Rhodamine B treatment improved MPS IIIA performance towards normal with treated mice having decreased escape latency, decreased incorrect entries and increased correct entries when compared to MPS IIIA untreated littermates. This provides the first report of SDT resulting in a beneficial effect on CNS function in an MPS disorder and SDT targeting gag synthesis may be a viable treatment option for children with MPS.

Single nucleotide polymorphism (SNP) microarray technology allows elucidation of a virtual karyotype at much greater resolution than traditional cytogenetic techniques. The size of deletions of a bi- or mono-allelic nature, clone size and concomitant abnormalities can be assessed in a single test. Is array therefore inherently better than traditional karyotyping? At what stage should it become the new standard of care? A case presentation for a patient with chronic lymphocytic leukaemia (CLL) will be presented to provide the evidence for advantages of SNP array over classical karyotyping. A summary of recent CLL cases analysed by SNP array from our centre will be discussed in terms of diagnostic and prognostic value, supporting the case that, at least for CLL, SNP arrays are ready to supplant classical karyotyping for diagnosis.

The ideal free distribution (IFD) theory is one of the most influential theories in evolutionary ecology. It predicts how animals ought to distribute themselves within a heterogeneous habitat in order to maximize lifetime fitness. We test the population level consequence of the IFD theory using 40-year worth data on pike ( Esox lucius ) living in a natural lake divided into two basins. We do so by employing empirically derived density-dependent survival, dispersal and fecundity functions in the estimation of basin-specific density-dependent fitness surfaces. The intersection of the fitness surfaces for the two basins is used for deriving expected spatial distributions of pike. Comparing the derived expected spatial distributions with 50 years data of the actual spatial distribution demonstrated that pike is ideal free distributed within the lake. In general, there was a net migration from the less productive north basin to the more productive south basin. However, a pike density-manipulation experiment imposing shifting pike density gradients between the two basins managed to switch the net migration direction and hence clearly demonstrated that the Windermere pike choose their habitat in an ideal free manner. Demonstration of ideal free habitat selection on an operational field scale like this has never been undertaken before.

New technology enables expansion of newborn screening (NBS) of inborn errors aimed to prevent adverse outcome. In conditions with a large share of asymptomatic phenotypes, the potential harm created by NBS must carefully be weighed against benefit. Policies vary throughout the United States, Australia, and Europe due to limited data on outcome and treatability of candidate screening conditions. We elaborated the rationale for decision making in 3-methylcrotonyl-coenzyme A (CoA) carboxylase deficiency (MCCD), which afflicts leucine catabolism, with reported outcomes ranging from asymptomatic to death. In Bavaria, we screened 677,852 neonates for 25 conditions, including MCCD, based on elevated concentrations of 3-hydroxyisovalerylcarnitine (3-HIVA-C). Genotypes of MCCA (MCCC1) and MCCB (MCCC2) were assessed in identified newborns, their relatives, and in individuals (n = 17) from other regions, and correlated to biochemical and clinical phenotypes. NBS revealed eight newborns and six relatives with MCCD, suggesting a higher frequency than previously assumed (1:84,700). We found a strikingly heterogeneous spectrum of 22 novel and eight reported mutations. Allelic variants were neither related to biochemical nor anamnestic data of our probands showing all asymptomatic or benign phenotypes. Comparative analysis of case reports with NBS data implied that only few individuals (< 10%) develop symptoms. In addition, none of the symptoms reported so far can clearly be attributed to MCCD. MCCD is a genetic condition with low clinical expressivity and penetrance. It largely represents as nondisease. So far, there are no genetic or biochemical markers that would identify the few individuals potentially at risk for harmful clinical expression. The low ratio of benefit to harm was pivotal to the decision to exclude MCCD from NBS in Germany. MCCD may be regarded as exemplary of the ongoing controversy arising from the inclusion of potentially asymptomatic conditions, which generates a psychological burden for afflicted families and a financial burden for health care systems.

The availability of therapies for lysosomal storage diseases (LSDs) and clear documentation from animal studies that optimal therapy depends on early diagnosis have set the scene for newborn screening for LSDs. The combined incidence of this group of conditions is approximately 1 in 7000, well within the feasible range for newborn screening programmes. The availability of multiplex technology has facilitated the technical aspects of initial screening. The scientific challenge is to predict disease severity early enough to influence choice of therapy. LSD screening is discussed from the point of view of the scientists, the families affected by these conditions, the community and clinicians.

To study the effectiveness of a specific national programme of enzyme replacement therapy (ERT) for patients with severe forms of Gaucher disease, a disorder of sphingolipid metabolism resulting from an inherited deficiency of the lysosomal enzyme beta-Glucocerebrosidase.Prospective analysis of data submitted at entry and every 6 months on therapy. The responses of haemoglobin (Hb) and platelet (plt) concentrations, liver and spleen volumes were assessed.Forty-eight patients were treated with ERT for a minimum of 6 months. Forty patients had Type 1 disease and eight had Type 3B. The age range was 1-70 years (median 24 years). Duration of therapy at the time of analysis was 6-114 months.Thirty-six per cent of patients started with a normal Hb increasing to 76% after 6 months. The mean improvement in Hb from baseline to the end of study period was 20 g/L, when the Hb was normal in 85% (41 patients). Thirty per cent of patients had a normal plt count at the start of therapy, with a more gradual increase in the count at 6 monthly intervals of 50, 91, 108 and 142% of starting value. Seventy-five per cent of patients had a normal plt count at the end of study. Spleen volumes reduced by a mean of 56% in 33 evaluable patients, and the liver by 27% in 30 of 38 evaluable patients. Eight patients had an increase in liver volume of 28%.Enzyme replacement therapy produced a spectrum of beneficial responses in patients with Gaucher disease, but all had some evidence of reversal of haematological complications and/or reduction in visceromegaly. Future analyses will examine the effect of therapy on bone disease, prepubertal growth and quality of life.

Both nuclear and mitochondrial DNA mutations can cause energy generation disorders. Respiratory chain complex I deficiency is the most common energy generation disorder and a frequent cause of infantile mitochondrial encephalopathies such as Leigh's disease and lethal infantile mitochondrial disease. Most such cases have been assumed to be caused by nuclear gene defects, but recently an increasing number have been shown to be caused by mutations in the mitochondrially encoded complex I subunit genes ND4, ND5, and ND6. We report the first four cases of infantile mitochondrial encephalopathies caused by mutations in the ND3 subunit gene. Three unrelated children have the same novel heteroplasmic mutation (T10158C), only the second mutation reported in ND3, and one has the previously identified T10191C mutation. Both mutations cause disproportionately greater reductions in enzyme activity than in the amount of fully assembled complex I, suggesting the ND3 subunit plays an unknown but important role in electron transport, proton pumping, or ubiquinone binding. Three cases appear to have a de novo mutation, with no mutation detected in maternal relatives. Mitochondrial DNA disease may be considerably more prevalent in the pediatric population than currently predicted and should be considered in patients with infantile mitochondrial encephalopathies and complex I deficiency.

Study Smith-Lemli-Opitz (SLO) syndrome is an autosomal recessive disorder of cholesterol synthesis due to deficiency in 7-dehydrocholesterol (7-DHC) reductase. SLO is associated with specific dysmorphology, microcephaly, syndactyly, cleft palate, growth retardation, genital ambiguity and intellectual disability with autistic features. The autistic features respond to cholesterol supplementation. Increased levels of 7-DHC are a marker for SLO and routinely used for testing of autistic children. Methods A total of 5 years of requests for 7-DHC testing at S A Pathology were audited (2008-2012). Data were broadly classified according to clinical indications, including isolated autism, developmental delay, seizure, dysmorphism, neurological, anatomic, genital or metabolic anomalies. Data were sub-classified as single or multiple clinical indication/s or no clinical information supplied. Results 809 tests were performed and 16 cases of SLO were detected. No cases of SLO were diagnosed based on an isolated clinical indication of autism or where no clinical information was supplied. Discussion Experience of several years of 7-DHC testing at SA Pathology indicates that autism as an isolated clinical trait is not associated with SLO. 7-DHC measurement should not be included in the testing panel when autism spectrum disorders are the only clinical indication.

Medium chain acyl-coenzyme A dehydrogenase (MCAD) deficiency classically presents as hypoketotic hypoglycemia. Under-production of ketones has been presumed to be the cause of hypoketosis, but this has never been proven. Stable isotope dilution studies of ketone kinetics were performed on three well children with homozygous 985G MCAD deficiency using 1,3-13C2 sodium acetoacetate and 1,2,3,4-13C4 sodium 3-hydroxybutyrate to ascertain the rates of ketone production, interconversion, and use. All children were fasted for 9 to 11.5 hours before the beginning of the study period. Euglycemia was maintained in all cases. Ketone kinetics were calculated using a two-accessible pool model and showed normal ketone production in all three children compared with published control data from children fasted for a similar length of time. There is no evidence for underproduction or overuse of ketones in these MCAD-deficient children, at least when they are well. We propose that another factor, such as fever, may be required to reduce ketone production and result in the biochemical phenotype recognized in unwell children.

We report two siblings with carbohydrate-deficient glycoprotein syndrome (CDG) type 1 (McKusick 212065) secondary to phosphomannomutase deficiency, both of whom have repeatedly normal transferrin isoform screening tests.

Background: Arginosuccinic acid synthetase (ASA) (EC 6.3.4.5) deficiency (citrullinaemia) (McKusick 215700) is a well-recognized cause of neonatal hyperammonaemic coma with poor long-term intellectual function, despite good medical management. Methods: Cadaveric hepatic transplantation was performed in a 12-year-old boy with citrullinaemia under poor biochemical control. Subsequent development of fulminant hepatic failure necessitated a second cadaveric transplant. Psychometric assessments before and after transplantation were performed using a variety of age-appropriate tests. Results: Normalization of plasma ammonium in our patient post transplantation has resulted in dramatic improvement in mental functioning and well-being and he now enjoys a normal diet. Psychometric assessment confirmed decline in his abilities prior to transplantation with particular post-transplantation improvement in perceptual organization and visuospatial abilities; these did not, however, return to normal. His family report considerable reduction in stress associated with the unpredictable nature of previous hyperammonaemic crises and recurrent hospitalization. Conclusions: Liver transplantation should be considered as an early therapeutic option in children with citrullinaemia to prevent ongoing cerebral insult associated with hyperammonaemia.

2-Octynoic acid was administered by intraperitoneal injection to fasted Sprague-Dawley rats in an attempt to simulate medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency. The resultant urine organic acid profile showed a mild dicarboxylic aciduria but lacked the glycine conjugates characteristic of MCAD deficiency. Further studies with infused 13 C 4 -3-hydroxybutyrate and 13 C 2 -acetoacetate demonstrated reduced ketone production in treated rats compared with control animals. Although plasma ketone body concentrations were low in treated rats, plasma free fatty acids were also low, thereby providing diminished substrate for ketone production. This is the reverse of the finding in children with MCAD deficiency, who have low levels of plasma ketones despite elevated free fatty acids. These animal studies were therefore not helpful in improving our understanding of ketone body kinetics in children with MCAD deficiency.

We report a family who experienced an unexplained neonatal death. Twelve years after the death, we retrospectively diagnosed multiple acyl-coenzyme A dehydrogenase deficiency by demonstrating an abnormal acyl-carnitine profile in the child's archived newborn screening card, using tandem mass spectrometry.

A. Poole, A. Deane, J. Magarey, S. O’Connor, S. Millington, J. Fletcher, A. Zaknic, M. Summers, M. Chapman