Nuovi strumenti diagnostici : NGS era

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1 Nuovi strumenti diagnostici : NGS era Achille Iolascon, MD, PhD Dept. Molecular Medicine and Medical Biotechnology, University Federico II, Naples, Italy achille.iolascon@unina.it

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3 madre padre figlio neonato figlia 7 anni Emoglobine principali A F S C A, M, alcune Hb instabili F S, D, G, Lepore C, E, A2, O Arab

4 Diagnosi post-natale e pre-natale 1) Prelievo di sangue dai villi coriali (Villocentesi) (VIII- X settimana di gestazione) 2) Prelievo di sangue fetale (Cordocentesi) (XVIII- XXII settimana)

5 Percent of patients Median TDT patient age has increased during the last 3 decades Age distribution of β thalassemia patients over several surveys North America 1973 (n=243) 1985 (n=303) Italy/Greec e (n=443) 1993 (n=271) (n=319) 2003 (n=170) Age (years) Vichinsky E P et al. Pediatrics 2005;116:e818-e825. Cross-sectional study; n=781.

6 Genetic Era: From DNA structure to Complete Sequence 1975-Southern blotting 1953-Discovery of double helix(watson and Crick) 1978-First Molecular Diagnosis 1985-Description of PCR technology Start of Human Genome Project 1990-First experiment of Gene Therapy: ADA deficiency correction 2000-First announcement of decodification of entire human genome

7 New DNA technologies Not only Diagnosis: predictive model - Predictive Prognosis - Predictive Therapy

8 Predictive model :

9 Era Pre-genomica Era Post-genomica

10 Time problem : diagnosis time 1 st 2 n Gene d Gene N Gene

11 Genomic testing strategies and clinical heterogeneity: pro and cons

12 Genetic heterogeneity increases as phenotypic specificity decreases

13 Diagnostic rates based on WES in classes of pediatric diseases

14 There is a balance between diagnostic potential of a sequencing strategy and its practicality and costs. Wright CF et al. Nat. Rev. 2018

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16 1 - Clinical Case: B. L. Male, 42 yrs Since the age of 2 yrs, diagnosis of microcytic anemia Cousins affected by Thalassemia Major HBA-HBB: b 0 heterozygous I1 I2 II1 II2 II3 I3 II4 I4 Analytics Value RBC 10*6/uL 4,91 Hb g/dl 10 HCT % 30,2 MCV fl 61,5 MCH pg 20,4 MCHC g/dl 33,1 RDW % 18,9 PLT 10*3/uL 279 Reticolociti % 8,50 Reticolociti 10*3/uL 417 WBC 10*3/uL 8,61 At 37 yrs: splenectomy (spleen 18 cm) and cholecystectomy (symptomatic litiasis), no improvement

17 B.L., 42 yrs He received > 30 transfusions IS>>75% - start chelation therapy Peripheral blood smear: target cells and several erythroblats, Analytics Value RBC 10*6/uL 3,55 Hb g/dl 7,4 HCT % 22,3 MCV fl 62,8 MCH pg 20,9 Analytics Value Reticolociti % 12,07 Reticolociti 10*3/uL 428 Bil totale mg/dl 7,6 Bil diretta mg/dl 1,4 Bil indiretta mg/dl 6,2 LDH U/L 1180 Aptoglobina undetectable MCHC g/dl 33,3 RDW % 17,6 PLT 10*3/uL 109 WBC 10*3/uL 4,3

18 B.L., 42 yrs Enzymatic Panel: Pyruvate kinase (PK) deficiency Genetic testing confirmed the presence of homozygous missense mutation in PKLR gene PK deficiency in a carrier of HBB mutation

19 The molecular data obtained by our t-ngs analysis confirmed the clinical suspicion in 54.2% of patients (26/48) (matched phenotype-genotype) The multi-gene approach modified the original diagnosis in 45.8% of patients (non-matched phenotype-genotype) 84.6% of patients with matched phenotype-genotype are included in the subgroup of RBC membrane defects 81.8% of non-matched patients were clinically suspected to suffer from CDA

20 Multi-gene panel testing improves diagnosis and management of patients with hereditary anemias Russo R, et al. Am J Hematol % of CDA patients within our cohort exhibited mutations in PKLR gene overlapping phenotypes among these disorders

21 3 - Clinical Case: E.C. Born after 38 weeks of normal pregnancy Apgar 8-9, AGA At 3 days: jaundice (bil 15.5 mg/dl), treated with photptherapy At 6 weeks: severe anemia Maternal fetal incompatibility, and G6PD deficiency were excluded Non-responder to EPO treatment Transfusion regimen (each 3 week) Congenital dyserythropoietic anemia?? Hereditary spherocytosis?? Analytics Value RBC 10*6/uL 1,44 Hb g/dl 4,75 HCT % 11,3 MCV fl 77,4 MCH pg 32,9 MCHC g/dl 42,3 RDW % 21,3 Retics % 7,98 Retics 10*3/uL 115 WBC 10*3/uL 14,2 PLT 10*3/uL 207

22 E.C., 6 weeks Congenital dyserythropoietic anemia?? No hypoglycosylation of band Hereditary spherocytosis?? Border line results of indirect tests Padre Madre Pink 13,2% 12% AGLT50 2h > AGLT50 2h > EMA test Normal range Border line 1. Proband 2. Proband 3. CTR 4. CTR +

23 E.C., 3.5 yrs I.1 I.2 II.1 II.2 SPTA1 LOCUS WT c.5029g>a, p.gly1677arg Alpha Lely c.2319c>a, p.cys773ter WT SEC23B LOCUS c.1254 T>G, p.ile418met WT GATA1 LOCUS g.4909g>a Hemolytic anemia due to poligenic inheritance

24 We herein analyzed a cohort of 123 HSt patients enrolled in our Genetic Unit (UOC University Hospital Federico II, Naples) from 2013 to We performed a descriptive statistics of the different subtypes among HSt. Moreover, we established a genotype-phenotype correlation on PIEZO1 mutated subjects useful for diagnosis, prognosis, and management of these patients

25 Genotype-phenotype correlation and risk stratification in a cohort of 123 hereditary stomatocytosis patients HSt class includes: 1. Dehydrated hereditary stomatocytoses type 1 and 2 (DHS1-2; PIEZO1/KCNN4) 2. Overhydrated hereditary stomatocytosis (OHS, RhAg) 3. Sitosterolemia/Phytosterolemia non leaky stomatocytosis (STLS; ABCG5/ABCG8) 4. Cryohydrocytosis (CHC; SLC4A1) 5. Familial Pseudohyperkalemia (FP; ABCB6) PIEZO1 resulted the most frequent mutated gene in our cohort of HSt patients ABCG5 6% SLC4A1 8% KCNN4 6% Negative 16% ABCB6 17% PIEZO1 47%

26 Bone marrow responsiveness index in PIEZO1 - patients DHS1 subjects showed lower reticulocyte count compared to those observed in other subgroups with similar Hb levels (KCNN4- and SLC4A1- patients) We observed lower BMRI values in DHS1 patients compared to those observed in HS ones This suggested that PIEZO1-patients might suffer from delayed erythrocyte maturation as previously hypothesized BMRI 400 P < P < CDAII (n=111) HS (n=25) DHS1 (n=16) DHS2 (n=3) Hb < 12.5 g/dl RBC Reticulocytes Erythroblasts RBC precursor

27 Genotype-phenotype correlation and risk stratification in a cohort of 123 hereditary stomatocytosis patients Our cohort of PIEZO1 patients was divided into two groups, severe and mild phenotype, using a W-Rank score that combines the ranking information obtained by several clinical variables Non- PORE PORE Patients showing a more severe phenotype carried mutations in the pore domain of the channel (77%) responsible of the ion passage and interactions, while patients showing a less severe phenotype carried mutations in the non-pore domain (75%), responsible for the mechanosensitive properties of the channel

28 Acknowledgements Lab n. 3 - CEINGE Medical Genetics Unit