Irene Valls Prez Master in Advanced Genetics UAB

Irene Valls Pérez Master in Advanced Genetics UAB

Introduction • The completion of the Human Genome Project in 2001 initiated a rapid expansion of knowledge about human DNA and genetic variation. • Sequencing technologies remained relatively expensive and time consuming, and clinical applications in subsequent years were mostly limited to the evaluation of rare monogenic Mendelian disorders via the Sanger method of DNA sequencing. • The development of massively parallel sequencing has accelerated the implementation of genomic sequencing (GS) in clinical practice. • The expanding knowledge base of associations between genetic variation and human disease signifies that the long-anticipated genomic revolution is underway.

Clinical genomics is the use of genome sequencing to inform patient, diagnosis and care. Genome sequencing is expected to have the most impact in three main areas: üCharacterising and diagnosing rare and inherited disease; üMolecular stratification of cancer to direct treatment pathways. üTo optimice drug choices and drug dosages based on an individual´s genotype (Pharmacogenomics).

• Bi-directional relationships: • Genomics Clinical Personal SNPs could be interpreted as mutations and thus indicate possible diseases/sensitivities. • Clinical Genomics Patient and family history leads to genetic testing order. Crosschecking of genomic results.

• The previous assessment to the genetic test • Health care along with the genetic Council determines if we are in a context of research or assistance. But of way immediate, taking in acount the costs and the availability of the technology the Center and the medical must set its order of priorities for offer these studies to them patients and agree well the use future of the information.

• • • 1. Purpose of the genetic analysis for which you consent. 2. Place of analysis. 3. Destination of the biological sample at the end of the same. 4. Persons who will have access to the results 5. Right to know the genetic data obtained 6. Warning about the possibility of unexpected findings and their possible transcendence for the subject. 7. Warning of the implication that can have for their relatives the information 8. Commitment to provide genetic counseling 9. Provision of international transfer of genetic data. 10. Possibility of identifying other persons exercising the right of access.

• The choice for the diagnosis of one of the NGS techniques must be determined by: • • the greater detection lower cost minor diagnostic time the retail information incidental and uncertain potential.

Two factors determine the choice of the technique of NGS to choose: • Rate potential detection technique for the person questioned phenotype • The frequency of finds uncertain or incidental little relevant of these. Panels of NGS • Exons and intronic flanking regions of a given gene number WES • Exons known as well as exon-intron flanking regions potentially involved in the splicing WGS • Chenges in no coding reagions

Sequence formats: Data not filtered or analyzed FASTQ BAM

Analisis level…filtered analyzed data • Extraction of genomic variants • Filtering variants • Identification of functional variants The most widely used format for storing genetic variant information is the Variant Calling Format (VCF)

In a genomic study we can find… • A) mutations that confirm the disease that caused the analysis of the patient; • B) mutations that are clinically relevant and whose knowledge is of direct benefit to the patient or his or her offspring (either because they can be intervened on them or because, although they do not have treatment, their knowledge can help the patient to make reproductive decisions); • C) high-penetrance mutations related to late-onset diseases (with and without current treatment); • D) mutations that do not cause disease in the patient but can cause it to their offspring (carriers of autosomal recessive diseases or carriers of diseases linked to the X chromosome) • E) mutations that confer a certain susceptibility to a particular complex disease, or variants and changes related to genetic traits of doubtful relationship with human health.

Information in the report: • The clinical reason of the request to answer the applicant to a specific question. • The type of study: diagnostic, predictive, prenatal, informative, pharmacogenetic, etc. • The methodology used, its sensitivity and specificity, the coverage, and other data that and reliability of the information. The results should include: All relevant data from the sequence analysis stratifying its clinic. The interpretation of these results in terms of clinical relevance The implication of the results in terms of medical management and genetic counseling.

Qualifed staff for a good management Assesment before and after the genetic study Knowledge in clinical and molecular genetics. Clinical geneticist …Need for training programs for professionals and the public and more specific formation for the staff…

• Future • Individualiced genomes are the future of individualiced care. • Population of genomes are the future of medical reseach There is no room for thecnophobes in the medical proffession. • As a doctor swipes her patient´s genetic ID card, the information downloads in seconds. • A string of letters scroll down her computer screen These are the raw data of DNA, the code of life It seems reasonable to imagine genome sequencing happening as a matter of routine in like 25 years time

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CASE EXAMPLE Making a definitive diagnosis: Successful clinical application of whole exome sequencing in a child with intractable inflammatory bowel disease.

• The patient is a male who initially presented at 15 months with poor weight gain and a perianal abscess. The abscess enlarged, drained spontaneously, but failed to close despite several rounds of oral, and then parenteral, antibiotics. • He subsequently developed diarrhea and weight loss. • He had a weight of 8. 1 kg, length 81. 2 cm, and body mass index of 12. 7, indicating severe stunting and malnutrition. Examination under anesthesia showed perineal fistulae and deep fissures. • Biopsy showed focal active proctitis with ulceration. • The child was treated with nasoenteric feeds and infiximab for a presumptive diagnosis of Crohn disease.

• Despite treatment, the perineal fistulae persisted, and new ones developed threatening the scrotum. . . Autoimmune disorders

• Immunologic and genetic evaluation for immune defects known. The hypothesis of an immune defect, the severity of the unchecked disease, and the significant long-term risks of the existing treatment regimen prompted discussion about the appropriateness of immune reconstitution. Identify the causative mutation, providing the necessary information for management, through exome sequencing.

This study was conducted under the oversight of an appropriate constituted institutional review board, and informed consent was obtained from all human subjects. Given the severity of the disease and the inability to make a diagnosis, the institutional review board approved the use of exome sequencing to facilitate a clinical diagnosis. Methods • Exome analyis… Roche 454 sequencing • Variant identification and annotation… Roche 454 gs. Mapper Sofware • Capillary sequencing validation…A small number of identified variants were validated in-house using ABI 3730 XL automated DNA Sanger sequencing • Actication-induced cell death • NOD signalling

Results Whole exome sequencing identified 16, 124 variants when compared with the human genome reference sequence; 14, 597 of these variants had previously been identified, and 1, 527 were novel. The remaining variant was a G to A substitution at a highly conserved position in the X-linked inhibitor of apoptosis gene (XIAP), resulting in a hemizygous cysteine to tyrosine amino acid substitution.

Phylogenetic conservation of the variant amino acid. A multiple sequence alignment of the region of the XIAP protein containing the variant. The cysteine is conserved in all species identified. The C-Y substitution is visible in blue at position 231.

Clinical confirmation in the child and mother. The region of the XIAP gene surrounding the mutation in both the child and the mother was sequenced using the Big. Dye Terminator Cycle Sequencing kit and analyzed on an ABI 3730 XL automated DNA sequencer. The Sanger sequence trace from a normal human control is shown at the top. Hemizygosity at the candidate locus is confirmed in the child (middle panel). The mother is heterozygous at this locus (bottom panel).

• The XIAP protein has a central role in the proinflammatory response, leading to activation of NFk. B and subsequent activation of proinflammatory cytokines via the NOD signaling pathway, as well as a crucial role in mediating programmed cell death.

• On the basis of data described in this report, this child has been diagnosed as having an XIAP mutation and resulting immunodeficiency. After a review of the findings, including the risks, benefits, and alternative treatments, an allogeneic hematopoietic progenitor cell transplant was recommended based on the mutation found in XIAP and performed in concordance with the suggested treatment for XIAP deficiency. At >42 days posttransplant, the child was able to eat and drink, and there has been no recurrence of GI disease, suggesting this mutation also drove the GI disease.