A novel 33-Gene targeted resequencing panel provides accurate, clinical-grade diagnosis and improves patient management for rare inherited anaemias

Accurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next-generation-sequencing (NGS) has been used to investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical-grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically-reliable diagnostic test and minimize false-negative results we developed an open-source tool (CoverMi) to accurately determine base-coverage and the ‘discoverability’ of known mutations for every sample. We validated our 33-gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS-based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family

Prenatal Diagnosis of Haemophilia - psychological, social and ethical aspects

The general aim of this study has been to study the psychological, social and ethical aspects for carriers of haemophilia and their spouses, related to the development of techniques for carrier testing and for prenatal diagnosis (PD) of haemophilia. This was investigated by a semi-structured follow-up interview with 29 carriers of haemophilia with experience of PD by fetal blood sampling (FBS), and with 29 carriers (and 23 spouses) with experience of PD by chorionic villus sampling (CVS). The long-term psychological effects of carrier testing, PD and abortion were investigated by a questionnaire study, comparing 105 carriers of haemophilia with and without experience of PD as well as a partially matched control group of 262 women who were not carriers of haemophilia and who had no experience of PD. Many carriers report negative emotional reactions when receiving information about carriership as well as a negative effect on their self image. Most aspects of the PD procedure by FBS were experienced as trying or very trying by somewhat less than half of the women. The prolonged period of uncertainty before the final results of the FBS process were obtained, implied that many carriers had not experienced the pregnancy as real until that point in pregnancy. For some of them even the remainder of the pregnancy became psychologically trying. PD by CVS was generally well accepted by the women and their spouses, appears to be experienced as less complicated than FBS and women do not report problems during the remainder of pregnancy. Positive effects on how the women experienced CVS resulting from participation by the spouse could not be identified. Carrier testing for haemophilia per se and in combination with PD does not appear to have negative long-term psychological effects among carriers who have children. Selective abortion after first trimester PD is experienced as emotionally painful by both the woman and her spouse, the reactions appearing similar to, but not as pronounced as, those found after second trimester selective abortion. Notably, signs of depressive mood were ob-served several years after the abortion, but negative long-term psychological effects could not be identified at a median of 6 years after the abortion

Parenting stress and its correlates in an infant mental health unit : a cross-sectional study

Background: An infant’s development is closely linked to the relationship they have with their parents. Parenting stress, affective disorder, and an upbringing with substance-abusing parents can affect parenting quality and increase the risk of children developing behavioral, mental and social problems. The overall aim of the study was to investigate how parents of children attending an outpatient Infant Mental Health (IMH) unit rate their own psychological health and parenting stress, and to explore predictors of parenting stress. Methods: The sample comprised 197 parents, 129 mothers and 68 fathers, referred with their infant/toddler to an outpatient IMH unit for interplay treatment. On admission, the parents completed self-report questionnaires concerning their own mental health problems and parenting stress. Results: The mothers reported significantly more psychiatric symptoms and parenting stress than the fathers. Fathers with substance-abusing parents had often experienced divorce in the family of origin, had a low level of education, and had often experienced trauma. Depression was a predictor for parenting stress for both mothers and fathers. Conclusion: The parents’ situation was strained, presenting a variety of psychiatric symptoms and high levels of parenting stress, making assessment of parental health before starting treatment important. The mothers’ situations were more serious compared with the fathers’, and for both parents depression was a significant predictor for parenting stress. To increase the chances of a positive treatment outcome for the child, both parents should be included in the treatment

Plastic surgical treatment of purpura fulminans: Long-term follow-up of two patients.

Abstract Purpura fulminans (PF) is a rapidly progressing, potentially life-threatening condition characterised by disseminated intravascular coagulation (DIC) and haemorrhagic infarction of the skin. Plastic surgical treatment of PF has never been reported in Scandinavia. The aim of this report was to review plastic surgical treatment of PF and the long-term results of two patients treated in our department. Both patients presented to a community hospital with skin lesions looking like simple traumatic skin bleeds a period after a Varicella infection. They were initially treated at the community hospitals with broad-spectrum antibiotics and adjunctive therapies. When their condition permitted, they were transferred to the department of paediatrics of Skåne University Hospital where their DIC was treated further. The patients were transferred to the department of plastic and reconstructive surgery, when medically stable, and operated on with debridement, and amputation of a toe in one patient, and the application of autologous skin grafts. The children made an excellent recovery and were discharged home after 1.5 months and 3 weeks, respectively. At follow-up, 14 years and 8 years later, respectively, the patients were fully recovered and no secondary corrections were indicated. In conclusion, debridement of necrotic tissue should be performed in a department of plastic and reconstructive surgery as soon as the child is clinically stable, and skin grafting when the wound bed permits it. Follow-up should be performed in the same fashion as for full-thickness burns

Genetic counselling of haemophilia carriers

Basic analysis of a potential carrier includes calculation of the probability, or odds, for carriership based on pedigree and clotting factor analysis. Genotype assessment constitutes a more accurate method of carrier detection. Where circumstances permit, the genetic diagnosis of hemophilia should be based on the direct identification of the pathogenic mutation in the factor (F) VIII gene. Neutral mutations in the FVIII gene and the risk of mosaicism (a mixture of normal and mutation carrying cells) in sporadic families may cause misclassification. If it is not possible to use the mutation for diagnostic purposes, it may be possible to use linked polymorphic markers (restriction fragment length polymorphisms [RFLP]) to trace the inheritance of the hemophilia gene within a pedigree. Linkage analysis is limited because of uninformative patterns of polymorphic markers, ethnic variation, linkage disequilibrium, and the need for participation of family members, and it is not useful in sporadic families, which constitute more than half of the hemophilia families. Potential carriers of hemophilia should be offered qualified assistance in genetic information, testing, and counseling to help them to cope with the psychological and ethical problems related to carriership of a genetic disorder

How do carriers of hemophilia experience prenatal diagnosis (PND)? : Carriers' Immediate and later reactions to amniocentesis and fetal blood sampling

A semistructured personal interview was performed with 29 carriers of hemophilia A or B, 1-5 years after a pregnancy in which prenatal diagnosis (PND) was performed by fetal blood sampling. Fetal blood sampling by fetoscopy was significantly more often reported by the women to the more trying than expected than was ultrasound-guided heart puncture. Of 29 women 13 was classified as having experienced the PND process (amniocentesis and fetal blood sampling) as distressing, having had mental or psychosomatic symptoms associated with it. All of the women who had abortion/miscarriage after PND reported a very high frequency of psychological sequelae during the 6 months that followed PND. Of 22 women who continued their pregnancy with a healthy fetus after PND 8 experienced the period until delivery as trying and felt that their emotional and somatic status influenced their daily life activities. This was particularly common among women who after fetoscopy received routine profylactic terbutalin treatment and had continuous sickleave until the 36th gestational week, 17/29 would consider going through PND in the future. Qualified psychological assistance must be offered both before and after PND

Prenatal diagnosis of haemophilia in Sweden now more commonly used for psychological preparation than termination of pregnancy.

The aims of the study were to define the frequency, outcome and reasons for prenatal diagnosis (PND) in Sweden during a 30-year period in order to study trends and changes. The study population, from the Swedish nationwide registry of PND of haemophilia, consisted of 54 women, compromising >95% of all, who underwent PND (n = 90) of haemophilia during 1977-2013. PND was performed by amniocentesis (n = 10), chorionic villus sampling (n = 64) or by analysis of foetal blood (n = 16). A total of 27/90 foetuses were found to have haemophilia. Sixteen went to termination and the remaining 11 were born during the end of the study period (2000-2013). Three of 90 pregnancies were terminated due to findings other than haemophilia and 3/90 PNDs led to miscarriage. In the 30 families with known haemophilia, PNDs (n = 55) were used in 27/55 cases for 'psychological preparation' and in 23/55 cases with the aim to terminate the pregnancy. A subgroup of women (n = 17) who consecutively underwent PND in the years 1997-2010 were further interviewed. For 11/17, being a carrier had a negative effect on the decision to become pregnant, and in 11 cases PND had influenced their decision to conceive. Our study show that PND of haemophilia is stable over time but increasingly used during the last decade as a psychological preparation for having a child with haemophilia as compared to earlier where more terminations of pregnancies were conducted

Various regimens for prophylactic treatment of patients with haemophilia

Haemophilia prophylaxis is superior to on-demand treatment to prevent joint damage. High-dose prophylaxis' as used in Sweden is more effective in preventing arthropathy than an intermediate-dose regimen' (the Netherlands) and the Canadian tailored primary prophylaxis. Prophylaxis may reduce the risk of developing inhibitors. There is no difference in inhibitor risk between plasma derived and recombinant factor VIII (rFVIII) products but the Rodin study showed increased risk with second-generation rFVIII products. MRI is a new and very sensitive tool to detect the symptoms of early arthropathy but some results (soft tissue changes in bleed-free joints') still need to be investigated. Ultrasound is a very helpful method to aid diagnosis especially during the acute phase of a bleed. The risk of infection with central venous access remains a matter of debate. A fully implanted central venous access device (CVAD) has a significant lower risk of infection compared to external CVADs. Patient's age under 6yr and inhibitor presence are additional risk factors for infections. The role of arteriovenous fistulae needs to be investigated because significant complications have been reported. Disease-specific quality of life instruments are complementary to generic instruments evaluating QoL in patients with haemophilia and have become important health outcome measures