Screen Positive Follow-up In The Clinic

Sherly Pardo-Reoyo, M.D.Medical Geneticist

Assistant Professor Dept. Biochemistry & PediatricsUPR-School of Medicine

Clinical Director MSMS and Molecular Laboratories PR-NBS Program

NBS Molecular Training Workshop April 2016

Newborn Screening (NBS) Goals

Ultimately the goals of newborn screening are to prevent the morbidity and mortality of infants by the early

identification of various diseases.

U.S. Dept of Health and Human Services.Discretionary Advisory Committee of Heritable Disorders in Newborns and Children.

U.S. Dept of Health and Human Services.Discretionary Advisory Committee of Heritable Disorders in Newborns and Children.

Success stories…

Newborn Screening (NBS) TIMELINESS

-Sample to be collected within 24-48 hours of life.

-Sample to be delivered to the NBS Lab within 24 hours of being collected.

-NBS tests to be completed and reported within day 5 of life for time-critical conditions and at least by the 7 day of life for all others.

Arizona, Colorado and Wyoming, California, Hawaii, Iowa, Michigan, Minnesota, Montana, Nebraska; Delaware, Maryland, New Jersey,

New York; Oklahoma, Puerto Rico, Tennessee, Texas, Virginia, Wisconsin

NBS…Beyond a dried blood spot.

www.gizmag.com

NBS Components

www1.newbornscreening.ph

Health care systems affect the TIMELINESS for prompt

confirmation and management.

Biochemical assay limitations

There is a differential diagnosis or set of disorders associated to a particular biochemical marker identified by NBS tests.

Most metabolic disorders are inherited in an autosomal recessive manner. Most patients do not have a null mutation causing 0% enzyme activity… thus levels of biochemical makers, age of onset, disease severity is variable.

Sometimes mild elevations in biochemical markers are due to maternal conditions or a partial deficiency in carriers who would not develop the disease.

Factors that affect a biochemical assay: age of infant at the time of sample collection, feeding status of infant at the time of sample collection, medications, population cut-offs.

SECOND TIER TESTSBIOCHEMICAL

MOLECULAR

Molecular tests are as good as it is their design: targeted, sequencing, rearrangement analysis.

-Allele drop-out (false-negative results)

Molecular test results are as good as the available evidence to determine their clinicalsignificance.

-Variants of uncertain significance (VUS)

-Pseudogenes: NOT all biochemical abnormalities (elevation of disease markers or even proven in vitro enzyme deficiencies) are associated with disease onset.

Many physicians and/or health professionals are not aware of these and might use or interpret the NBS or any molecular test results inappropriately.

Molecular assay limitations

Journal Sentinel

Your screen is positive on a 5 day old infant … now what?

The clock is ticking…Toxic substances and/or secondary deficiencies are building up and causing damage… some irreversible.

Diagnostic GuidelinesFor many genetic conditions, MOLECULAR TESTS are enough to establish the diagnosis without the need for active disease manifestations or further tests.

A few examples are:-Marfan Syndrome

-Neurofibromatosis Type 1

-Duchenne/Becker Muscular Dystrophy

-Cystic Fibrosis

For many others it is still recommended to conduct MOLECULAR TESTS to guide management and for prognosis.

Do not forget there are various mechanisms that result in mutations/variations.

Examples of tests and mutation types in NBS screened disorders.

CASES FOR DISCUSSIONStudy Case #1

Cystic Fibrosis

Study Case #1

Baby boy #2 is a 35 week old born by cesarean section due to failure to progress after 14 hours in labor. The infant was born lethargic with significant aspiration of meconium. He was stabilized and sent to the neonatal intensive care unit with ventilatory support and placed on total parenteral nutrition. The newborn screening sample was obtained at 40 hours of age and sent to the NBS laboratory within 24 hours of collection. On day of life 4 the infant is still lethargic and a sepsis work up was started. Meconium was passed on day of life 1. On the second day of life a Genetics consult was placed since the infant has a heart defect and some unusual features.

On day of life 5 the NBS Lab calls in since the CF screening test was positive. They report that the infant’s IRT level was elevated. They perform targeted molecular tests for certain mutations and they found one, F508del.

The family history reveals that the parents are first cousins. They just arrived from Asia and they do not have medical insurance.

What to do next?Option 1:You decide to send a second sample to the NBS laboratory for confirmation.

Option 2:You decide to refer to a CF Center for sweat testing once the infant is discharged.

Option 3:You decide to send for full sequencing of the gene prior to sweat testing..

All assays including NBS tests have their limitations. You should know them before making your follow-up and/or re-testing algorithm.

CF Clinical features• Obstruction of tubular structures by viscous secretions.

Respiratory system

en.wikipedia.org

www.emaze.com

Gastrointestinal system

www.pinterest.com

Sweat test: $25-100Gene sequencing: >$1000

CF Professional Guidelines

CF Professional Guidelines cont.

CFTR mutation/variation databases: CFTR1 and CFTR2

CFTR Genotype 1

Range of Phenotypes 2First Allele

Second Allele(or Homozygous)

Classic (e.g., ΔF508) Classic Classic >> non-classic

Mild (e.g., A455E) Classic or mild Non-classic > classic

R117H/5T Classic or mild Non-classic > classic

R117H/7T Classic or mildAsymptomatic female or CAVD > non-classic

5T/TG13 or TG12 Classic or mildCAVD or non-classic CF >> asymptomatic carrier

5T/TG11 Classic or mild Asymptomatic > CAVD

7T or 9T Classic or mild Asymptomatic

7T or 9T 7T or 9T Asymptomatic

Table 4. Genotype-Phenotype Correlations: Cystic fibrosiswww.genereviews.org

Clinical Effects of Various CFTR Mutation Combinations

WOULD THE MOLECULAR TEST AFFECT THE MANAGEMENT OF A CF PATIENT?

Kalydeco (ivacaftor), 150 mg po bid, Is a FDA approved prescription medicine used for the treatment of cystic fibrosis (CF) in patients age 6 years and older who have one of the following mutations in their CFTR gene: G551D, G1244E, G1349D, G178R, G551S, S1251N, S1255P, S549N, or S549R.

KALYDECO was granted Breakthrough Therapy designation by the U.S. FDA in late 2012 and FDA approval in January 21, 2014.

Ivacaftor facilitates increased Cl- transport by potentiating the channel-open probability (Class III mutations).

G551D-CFTR protein defectwithout KALYDECO

G551D-CFTR protein defectwith KALYDECO

www.kalydeco.com

Precision medicine/ Genotype-based management: CF

Kalydeco costs ~$290,000 per year per patient.

Adults and children 12 years and older should take 2 tablets by

mouth every 12 hours. The wholesale acquisition cost per

dose (2 tablets) is $354.80, equating to an annual cost of

$259,000 per year per patient.Jennifer L. Cruz, PharmD, BCPS, and Katherine Summers,

PharmD candidatePublished Online: Friday, November 20, 2015//

http://www.pharmacytimes.com

DO WE KNOW ALL ABOUT CFTRMUTATIONS?

CFTR mutations have been well studied for years. Do we already know everything about them?

ClinVar Database: CFTR

What to do next?Option 1:You decide to send a second sample to the NBS laboratory for confirmation.

Option 2:You decide to refer to a CF Center for sweat testing once the infant is discharged.

Option 3:The infant might stay in the NICU for several weeks. Therefore you decide to send for full sequencing of the gene prior to sweat testing..

All assays including NBS tests have their limitations. You should know them before making your follow-up and/or re-testing algorithm.

CASES FOR DISCUSSIONStudy Case #2

Baby boy #1 is a 35 week old born by cesarean section due to failure to progress after 14 hours in labor. The infant was born lethargic with significant aspiration of meconium. He was stabilized and sent to the neonatal intensive care unit with respiratory support and placed on intravenous fluids. The newborn screening sample was obtained at 28 hours of age and sent to the NBS laboratory within 24 hours of collection. On day 3 of life the infant was still lethargic requiring ventilatory support and his nutrition was changed to total parenteral nutrition and intralipids.

On day of life 5 the NBS Lab calls in since the PKU screening test was positive. They report that the infants level of phenylalanine was 5 mg/dl and the population cut off is 2 mg/dl. They perform targeted molecular tests for certain mutations and they found one, R408W.

The family history reveals that the parents are first cousins. They just arrived from Ireland and they do not have medical insurance.

Study Case 2

Hyperphenylalaninemia

What to do next?Option 1:You don’t do anything because the levels were mildly elevated and since there was only one mutation identified he is most likely a carrier.

Option 3:You start a PKU diet/treatment ASAP.

Option 2:You ask for a second sample to repeat the NBS test.

PKU Professional Guidelines

PKU Professional Guidelines cont.

Thyroxine

Melanins

TyrosineHydroxylase

phedup.co.uk/images/untreated-pku.jpg

Phenylalanine metabolism

WOULD THE MOLECULAR TEST AFFECT THE MANAGEMENT OF A PATIENT WITH HYPERPHENYLALANINEMIA?

Precision medicine/ Genotype-based management:Hyperphenylalaninemia

Plasma amino acids (PAA): >$230Urine organic acids (UOA): >$230Gene Sequencing: >$1000Pterins: ~$700

Diagnostic work up.

DO WE KNOW ALL ABOUT PAH GENE MUTATIONS?

ClinVar Database: PAH

What to do next?Option 1:You don’t do anything because the levels were mildly elevated and since there was only one mutation identified he is most likely a carrier.

Option 3:You start a PKU formula/treatment ASAP.

Option 2:You ask for a second sample to repeat the NBS test.

CAN WE START THE CORRESPONDING BREAKTHROUGH MANAGEMENT AND THEN DISCONTINUE IT IF NOT NEEDED?

“As you can see, at current pricing, the cost of Naglazyme will pass $1 million as patients hit their teen years, and reach $1.3 million as they approach their 40s. Of course, it’s not certain that this will happen; one could imagine that the price may need to drop over the coming decades. But this shows how very expensive these drugs are”.

WHAT IS THE DIFFERENCE BETWEEN BIOCHEMICAL OR MOLECULAR GENETIC TEST REPORTS AND OTHER CLINICAL TEST REPORTS?

Consensus guidelines for sequencing reports.

Use the term “variant” with the following modifiers:

(i) pathogenic(ii) likely pathogenic (iii) uncertain significance (VUS)(iv) likely benign(v) benign

Consensus guidelines for sequencing reports cont.

A. Use of standardized terminology and established databases for reporting sequence variants1. Nomenclature. A uniform nomenclature is recommended to ensure the unambiguous designation of a variant and enable effective sharing and downstream use of genomic information. A standard gene variant nomenclature (http://www.hgvs.org/mutnomen) is maintained and versioned by the Human Genome Variation Society (HGVS).2. Reference sequenceA standard reference sequence for each gene should be used and derived from the NCBI

refseq database. The origin of the transcript used should be specified, e.g., the most common human transcript, largest known transcript, or tissue-specific alternatively spliced transcript.3. DatabasesSequence databases. Disease-specific databases. Population databases. However, disease and gene-specific databases often contain variants that are incorrectly classified. Specify:-The frequency with which the database is curated and the latest update;-Evidence for variant classification (it is recommended to check the original cited reference);-Number of times the variant has been reported;-Populations in which a variant has been reported.

Consensus guidelines for sequencing reports cont.

Human mutation/variation registries.

How to determine the clinical significance of VUS?

THE MOLECULAR ASSAY IS AS GOOD AS EACH OF ITS COMPONENTS: THE DESIGN, VALIDATION, ANALYSIS, BIOINFORMATICS.

OMIM

c.695A>G...... It is not on exon 5 of PAH, and does not result in R158Q

Design and analysis: do not believe the first reference you find.

PERSONAL EXAMPLES TO EXERT CAUTION WITH THE INTERPRETATION OF MOLECULAR RESULTS.

Amino acid (R) 158 is cDNApositions 472-473-474

c.695A>G is actually Q232R

c.1155C>G; Exon 11

Design and analysis: PAH discrepancies in gDNA and cDNA NCBI reference sequences.

OTHER EXAMPLES TO EXERT CAUTION WHEN INTERPRETING MOLECULAR TESTS RESULTS.

PALB2 gene:~45,000 nucleotides1186 amino acids

Reported as pathogenic mutations: 12 (as of Dic-8-2014)138 (as of May-12-2015), 37 by multiple submitters

Reported as pathogenic mutations: 1256 total419 multiple submitters (as of May-12-2015)

ClinVar: Examples of how rapidly the data in the registry changes.

BRCA2 gene:~89,000 nucleotides3418 amino acids

Discrepancy among protein predictive

models.

Optional supplementary reports

Discrepancy in the reference sequences used by laboratories and mutation registries.

Things to remember…

-Allele drop out can occur.-Bioinformatics can add bias.-Clinical mutations databases are not complete or the classifications are not always accurate.-There can be discrepancies on reference sequences and protein prediction models.-Most physicians who will receive the data/results are not molecularly savvy/competent.-NOT ALL molecular methods identify all possible genomic defects. A number of genomic alterations are due to rearrangements often missed on various molecular methods used regularly for identifying missense mutations. [Sanger Sequencing: >$1000; Del/Duplication analysis: >$700]

Summary.

• Molecular tests can be useful for screening, diagnosis, and prognosis.

• Remember that molecular tests have limitations. [Physicians must follow up the patients, if some manifestations develop then consider further molecular testing including del/dup analysis].

• Further education of healthcare professionals and/or Genetic Counseling regarding the need, utility and results interpretation for various molecular tests is necessary.

• Mutation/variant registries as well as natural history studies are necessary.