Whole Exome Sequencing in Inherited Endocrine Disorders

Whole Exome Sequencing in Inherited Endocrine Disorders Background Molecular diagnosis is important in the management of various paediatric endocrine disorders including disorders of growth, metabolism, bone, hypoglycaemia and sexual development. Traditional PCR-based Sanger sequencing is the mainstay format for molecular testing in paediatric cases. However, the large number of gene defects associated with the various endocrine disorders renders gene-by-gene testing increasingly expensive and unattractive. The large number of potentially relevant genes makes it challenging for hospital molecular diagnostic laboratories to offer gene-based testing of all candidates. Given the high costs associated with single-gene tests, the selection of candidates for single-gene sequencing tends to be sequential rather than inclusive and parallel. In practice, different genes may be outsourced to different clinical or in some cases academic research laboratories which adds to the complexity. Using new high-throughput sequencing technologies, whole genomes, whole e xomes or candidate-gene panels (targeted gene sequencing) can now be cost-effectively sequenced for endocrine patients. In the near future, protocols involving next-generation sequencing would probably be considered as an appropriate component of routine clinical diagnosis for relevant patients. Defects of pituitary hormones lead to abnormalities in growth (e.g., short stature), sexual development, fertility, stress response and other metabolic processes. A number of genes coding for transcription factors have been identified, mutations in which cause medical disorders in humans associated with pituitary deficiencies [1-2]. Some of these factors, such as PROP1, TPIT, POU1F1, LHX3 and LHX4, play roles in the normal embryological development of the anterior pituitary. Mutations in these genes can lead to multiple pituitary hormone deficiencies and/or syndromic hypopituitarism [3]. The transcription factors such as HESX1, OTX2, SHH, SOX2 and SOX3 are involved in midline development. Mutations in these can cause septo-optic dysplasia or holoprosencephaly, both of which may include pituitary hormone deficiencies [4]. Other genes encode the precursors to pituitary hormones (growth hormone, ACTH [through processing of POMC], gonadotropic-luteinizing hormone and follicle-stimulating hormone, and thyroid-stimulating hormone). Mutations in these genes lead to phenotypes characteristic of individual hormone deficiency. The pituitary secretory cells themselves respond to signals originating in the hypothalamus, some of which are also peptide hormones with specific receptors expressed on the responding cells; mutations in these genes or their cognate receptors can also cause combined or specific pituitary deficiencies [1]. However, many cases of congenital hypopituitarism still remain unexplained and most are presumably due to other causes, either mutations in other deve lopmental genes or epigenetic influences during embryogenesis. Short stature is a common presentation to the paediatric endocrinology clinics. However, no cause is identified in a large proportion of patients who are classified as having idiopathic short stature [5, 6, 7]. It is estimated that the underlying cause for short stature remains unknown in approximately 80% of patients [8]. In a large-scale pooled Next-Generation Sequencing study to identify genetic causes of short stature, 4928 genetic variants in 1077 genes were present in patients but not in control subjects [9]. Large-scale sequencing efforts have the potential to rapidly identify genetic aetiologies of short stature. In another study, seeking to identify known and genetic causes of short stature by conducting whole exome sequencing of the patients with severe short stature and their family members, genetic cause of short stature was found in 5 out of the 14 recruited patients [10]. Rare genetic defects in the GH/IGF-1 axis have been found to cause short stature. A higher frequenc y of rare CNVs (common number variants) has been reported in patients with short stature [8, 11]. A recent study to define genetic characterisation of a cohort of children clinically labelled as Growth Hormone or IGF1 insensitive found that whole exome sequencing contributed to the diagnosis of children with suspected growth hormone and IGF1 insensitivity, particularly in the Growth hormone insensitive subjects with low serum IGF1 SDS and height SDS [12]. It may be now possible to identify likely genetic causes of short stature by implementing genomic investigative techniques like whole exome sequencing in many of these children who have unknown reasons for their poor linear growth. Congenital Hyperinsulinism (CHI) is the most common cause of persistent and recurrent hypoglycaemia in infancy [13]. It is the result of unregulated insulin secretion from the pancreatic ÃŽ ²-cells leading to severe hypoglycaemia [13, 14]. This condition has been reported in nearly all major ethnic groups and affects at least 1/50,000 children of European descent [14]. CHI is caused by genetic defects in key genes regulating insulin secretion. The genetic basis of CHI involves mutations in nine different genes (ABCC8, KCNJ11, GLUD1, GCK, HADH, SLC16A1, HNF4A, HNF1A and UCP2), which regulate insulin secretion from the pancreatic ÃŽ ²-cells [14,15]. The most common molecular cause of CHI is the dysfunction of the pancreatic KATP channel encoded by the sulfonylurea receptor gene (ABCC8) and the inward rectifying potassium channel gene (KCNJ11) [14,15]. CHI can also be secondary to risk factors like birth asphyxia, intra-uterine growth retardation, Rh isoimmunisation and maternal diabetes mellitus or associated with various developmental syndromes [16]. Histologically, CHI can be associated either with diffuse insulin secretion or with focal adenomatous hyperplasia. Positron emission tomography scan using Fluorine-18 L-3, 4-dihydroxyphenylalanine (18-fluoro DOPA-TC-PET-scan) has been used to distinguish focal from diffuse forms. Medical treatments of CHI include diazoxide (KATP channel activator), somatostatin analogue (octreotide) injections, and appropriate diet. The surgical treatment with subtotal pancreatectomy is required in diffuse CHI when medical treatment and dietary therapies are ineffective, whereas focal CHI can be cured with resection of the focal area of adenomatous hyperplasia [14, 15, 16]. Recently, mammalian target of rapamycin (mTOR) inhibitor sirolimus has been used in treatment of persistent severe CHI not amena ble to medical therapies [18]. CHI has been described as an associated finding in various syndromes like Beckwith-Wiedemann, Kabuki, Trisomy 13, Mosaic Turner, Sotos, Usher, Timothy, Costello, Central Hypoventilation syndrome and Leprechaunism (Insulin Resistance Syndrome) [17]. However, in many patients, with clinically defined syndromic features and with hypoglycaemia, no identifiable genetic cause contributing to hyperinsulinism is found. In a large series of 300 patients, genetic diagnosis was made only in 45.3% of the patients and mutations in ABCC8 were the commonest identifiable cause [19]. The vast majority of patients with Diazoxide-responsive CHI (77.6%) had no identifiable mutations, suggesting other genetic mechanisms [19]. Molecular diagnosis can be very important for clinicians to manage the patients more effectively and to counsel parents on the prognosis and disease recurrence. Whole Exome sequencing can be advantageous in these groups of patients to identify the mol ecular defects and to assess the coding variants that may be pathogenic in these patients [20]. Aims To identify novel genetic causes of rare inherited endocrine disorders in children with a focus on congenital hyperinsulinism, short stature of unknown etiology and IGF1 abnormalities by using whole exome sequencing. Experimental Design and Methods Patient Recruitment Patients with a diagnosis of CHI referred to Alder Hey Children’s Hospital, which is a national referral centre for CHI, will be recruited into the study. A written informed parental consent will be obtained. These patients will be biochemically confirmed as CHI using the following criteria: Blood glucose concentration of less than 3.0 mmol/l with detectable insulin and/or C-peptide Glucose requirement > 8mg/kg/min Low levels of ketones and fatty acids during the episode of hypoglycaemia Clinical and biochemical data will be collated from referral letter or by case note review. Patients with a secondary cause of CHI such as perinatal asphyxia, intra-uterine growth restriction, Rhesus isoimmunisation, infants of diabetic mothers and infants with Beckwith Wiedemann syndrome will be excluded from the study. Patients are considered to be unresponsive to medical treatment if recurrent hypoglycaemia episodes ( Patients attending the Paediatric Endocrinology clinic at Alder Hey Children’s Hospital with severe short stature (>3 SDS below mean) for age and sex in whom the standard clinical work up has not revealed a diagnosis for their short stature will be recruited into the study. Patients referred or evaluated for growth hormone insensitivity (growth failure, low serum IGF1 and normal/elevated serum GH) or IGF1 insensitivity (pre- and postnatal growth failure associated with relatively high IGF1 levels) will also be recruited into the study. A written parental informed consent will be obtained prior to the recruitment. Whole Exome Sequencing (WES) WES will be performed at the Centre for Genomic Research (CGR) based at the University of Liverpool. The test will be ordered after explaining the risks and benefits of testing to the patient and obtaining written informed consent. Each patient (and their parents or guardians) will be advised of the potential disclosure of conditions unrelated to the indication for testing that might warrant treatment or additional medical surveillance for the patient and possibly other family members. Peripheral-blood samples will be obtained to isolate DNA from the patient and both parents where possible. High-throughput sequencing will be performed using Illumina HiSeq2500. The genomic DNA samples from probands will be fragmented, ligated to Illumina multiplexing and amplified by means of a polymerase-chain-reaction assay with the use of primers with sequencing barcodes. Variants that were deemed clinically significant will be confirmed by means of Sanger sequencing. Parental samples, if available, will also be analysed by means of Sanger sequencing. Further functional analysis will be undertaken to establish the pathogenicity of the identified variant by utilising the in-house lab facilities at Institute of Child Health. Relevance of the proposed project Despite the advances in understanding the molecular pathogenesis, specific genetic determinants are not known in nearly 50% of patients with CHI and 80% of children with short stature. Whole exome sequencing in this group of patients will help to understand and identify the potential causative mutations in genes implicated in insulin regulation and growth. This will help the clinicians to provide optimal treatment and to counsel patients on disease progression and recurrence risk. Identification of novel genetic aetiology has the potential to identify novel therapeutic strategies for these patients. The applicant will spend time initially at Alder Hey Children’s Hospital to recruit patients and then at Cincinnati Children’s Hospital to learn the techniques of WES, bioinformatics and functional analysis. The applicant will then return to Alder Hey Children’s Hospital Institute of Child Health, University of Liverpool to complete the study and strengthen the local research expertise relevant to next generation sequencing.

Ethics Audit Question Essay

Board – Does the board have a clear and concise plan of action to deal with problems that arise from regular assessment? This question is important when assessing an organization’s ethical performance. This question will help the organization create a plan of action to deal with any problem that may arise. Staff- Are all staff members educated equally about how to advance in the organization? This question would help the organization make sure that they are treating all employees the same, as well as giving them all the same opportunities. The question would also aid in the assessment of the organizations ethical performance. Donors and Fundraisers- Does the organization give the donors the proper instructions or information on how to write their donations as tax write offs or deductions? In an ethical performance review, the organization will show that it cares about the donors, and is following government procedures. Clients/Customers- Is there a policy in place to ensure that all clients are given the same amount of time to be helped and processed? In an ethical review this shows that all clients are treated equally without prejudice or bias regardless of age, ethnicity, race, origin, or sex. Volunteers- Are all volunteers treated the same, and given equal opportunities for employment in the organization? It is important for the organization to show their volunteers that they are valued and important to the organization. Society- Is the organization doing everything it can, to meet the expectations of the public it serves? It is important for the organizations ethical committee to know that the organization is doing all that it can to serve the public’s needs. The public will feel like they can trust the organization.

The Human Resource ( Hr ) - 1655 Words

The human resource (HR) in an organization deals with the day to day operations of the human resources department. The HR department deals with business law, compensation, employee relations, benefits, medical and the like. HR focuses on whom the organization hires, whom the organization fires and remediation to employees who need discipline and retooling to continue their employment. The functions of the HR department in my organization include: recruiting and retaining talent, performance management and compensation, employee benefits, recruitment and staffing and employee rights and safety. In spite of the turbulent environment facing the healthcare industry today, my organization has however proven its effectiveness in performance†¦show more content†¦Recruiting and retaining talent has been noted as a top priority for participating best-practice companies. Benson noted that attracting and retaining top talent personnel ties in with the belief that â€Å"getting the rig ht people on the bus is likely to lead to better employee performance and long-term business success†. Investing in training and organizational programs enhances employee skills and provide opportunities of future career growth to internal candidates. Leadership training is also required of newly hired and promoted supervisors and managers on topics such as performance management and how to handle employee relations matters at the department level. Mayhew and Media p 4 Employee benefits The HR department communicate effectively with employees and keep them informed of major issues and initiatives like sharing financial salary, and performance information to develop a high-trust organization with employees. They also provide employment security for employees who perform well so that they are not downsized because of economic downturns or strategic errors by senior management. With regards to compensation and benefits the HR functions include setting compensation structures and evaluating competitive pay practices. Mayew and

Decision Making An Integrated Structure Of Events

There are certain personality traits that team leaders require in order to assert influence and function properly in their role. As information and knowledge are disseminated throughout a team, the intellectual capacity of both the team and the organization is increased. Such a strategy is sometimes referred to as pyramiding or pyramid learning, meaning that knowledge is spread from one team member to another before eventually spreading throughout the entire team. Decision-making Decision making is an integrated structure of events that includes collecting, understanding, and exchanging information (). Pre-established measures and veto policies are vital when it comes to an organization developing strategies to aid in decision making†¦show more content†¦Team Motivation Employees that feel empowered by their leaders have greater job satisfaction, show commitment to the organization and generally have high performance are more creatively engaged. Motivation is the key to holding a team together. In many circumstances, motivation determines the amount of effort individuals are willing to put into their work. Therefore, it is important for you to ensure that your team is highly motivated in its work. Meyers Brigg indicator assist with this and furthermore can be linked into different motivation theories. By understanding and how MBTI are linked to motivation, helps to enhance the understanding the role in personality and particularly with the knowledge based personality processes. Individual can be either intrinsic or extrinsic motivated, however, knowing if your team members like to be motivated by rewards or by activities reflects back in a more productive and successful team. A lack of motivation in any team member can have a negative effect, not o nly reducing the team s effectiveness as a whole but also possibly leading to the demotivation of others. 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