1

Preconceptional Test For Monogenic

Diseases

Dr. José A. Horcajadas Associate Professor, U. Pablo de Olavide, Seville, Spain

Research Associate Professor, EVMS, Norfolk, Virginia, US CSO, Recombine Europe, Barcelona, Spain

The Human Genome Project started in 1990

² The Mission of the HGP: The quest to

understand the human genome and

the role it plays in both health and

disease.

“The true payoff from the HGP will be the ability to

better diagnose, treat, and prevent disease.” --- Francis Collins, Director of the HGP

and the National Human Genome

Research Institute (NHGRI)

1985-Human Genome Project 1985 2003

FINISHED!!! Lord, They discover the

complete Human Genome code!

Heavens!

Those … hackers!

I have to change

the password!

HUMAN GENOME PROJECT

+

NEW TECHNOLOGIES

=

THE “OMICS” REVOLUTION

Quackenbush 2006 N Engl J Med 354:2463-72

TRADITIONAL VERSUS OMICS APPROACHES

The Basic Paradigm of the Biology

PRECONCEPTIONAL

PGD/PGS PRENATAL

Complexity  of  the  Human  Genome    

•  3.500  million  pairs  of  bases  

•  Around  35.000  genes  

•  3%  are  genes,  97%  is  “junk”  DNA  with  some  regulatory  

acEvity  over  those  genes  (not  anymore  junk!)  

•  We  all  share  >99%  of  geneEc  informaEon,  with  >20  million  

pairs  of  bases  (SNPs)  being  different.  

GENETIC  BASIS  OF  DISEASE  

•  SNPs  (single  nucleoEde  

polymorphisms)  are  one  

base  change  (A,T,C,G)  in  the  

DNA  sequence.  

 

•  SNPs  explain  many  geneEc  

diseases  and  differences  

between  humans.  

•  Close  to  20M  SNPs  

idenEfied  

SNPs  

§  6000-­‐7000  single  gene  disorders  

§  Combined  incidence:    1/300  births  (U.S.)      

§  Everyone   carries   severe   recessive  

mutaJons  that  can  cause  geneEc  condiEons  

§  Recessive  mutaEons  can  pass  down  quietly  

through  many  generaEons    

§  Carriers  may   not   have   a   family   history   or  

symptoms  of  a  geneEc  disease  

§  Hence,   carrier   tesJng   is   the   only   way   to  

determine  carrier  status    

Autosomal Recessive

Inheritance

THE  BURDEN  OF  GENETIC  DISEASE  

1.  Bell,  C.J.,  Dinwiddie,  D.L.,  Miller,  N.A.,  Hateley,  S.L.,  Ganusova,  E.E.,  Mudge,  J.,  Langley,  R.J.,  Zhang,  L.,  Lee,  C.C.,  Schilkey,  F.D.,  Sheth,  V.,  Woodward,  J.E.,  Peckham,  H.E.,  Schroth,  G.P.,  Kim,  R.W.,  Kingsmore,  S.F.,  2011.  

Carrier  tesEng  for  severe  childhood  recessive  diseases  by  next-­‐generaEon  sequencing.  Sci  Transl  Med.  Jan  12;3(65):65ra4  

2.  Berry,  R.J.,  Buehler,  J.W.,  Strauss,  L.T.,  Hogue,  C.J.,  Smith,  J.C.,  1987.  Birth  weight-­‐specific  infant  mortality  due  to  congenital  anomalies,  1960  and  1980.  Public  Health  Rep.  102,  171–181.  

3.  Costa,  T.,  Scriver,  C.R.,  Childs,  B.,  1985.  The  effect  of  Mendelian  disease  on  human  health:  a  measurement.  Am.  J.  Med.  Genet.  21,  231–242.  

4.  Gukmacher,  A.E.,  Collins,  F.S.,  2002.  Genomic  medicine:  a  primer.  N.  Engl.  J.  Med.  347,  1512–1520.  

5.  Kingsmore,  S.,  2012.  Comprehensive  carrier  screening  and  molecular  diagnosEc  tesEng  for  recessive  childhood  diseases.  PLoS  Curr.  May  2:e4f9877ab8ffa9.  

6.  Kumar,  P.,  Radhakrishnan,  J.,  Chowdhary,  M.A.,  Giampietro,  P.F.,  2001.  Prevalence  and  pakerns  of  presentaEon  of  geneEc  disorders  in  a  pediatric  emergency  department.  Mayo  Clin.  Proc.  76,  777–783.  

7.  Scriver,  C.R.,  Neal,  J.L.,  Saginur,  R.,  Clow,  A.,  1973.  The  frequency  of  geneEc  disease  and  congenital  malformaEon  among  paEents  in  a  pediatric  hospital.  Can.  Med.  Assoc.  J.  108,  1111–1115.  

8.  Srinivasan,  B.S.,  Evans,  E.A.,  Flannick,  J.,  Pakerson,  A.S.,  Chang,  C.C.,  Pham,  T.,  Young,  S.,  Kaushal,  A.,  Lee,  J.,  Jacobson,  J.L.,  Patrizio  P.,  2010.  A  universal  carrier  test  for  the  long  tail  of  Mendelian  disease.  Reprod  

Biomed  Online.  Oct;21(4):537-­‐51.  

20%

infant deaths

PaEents  affected  by  rare  disorders  face  mulEple  

challenges  (survey  by  Eurodis  2006):  

•  40%  iniEally  receive  incorrect  diagnosis  

•  DiagnosEc  delay  (5-­‐30  years  for  25%  of  paEents)  

•  10%  received  Psychological  care:    

                       Wrong  assumpEon  of  psychosomaEc  basis  

•  33%  received  inappropriate  treatment:    

                       16%  unnecessary  surgery  

GENETIC  DISORDERS  AND  SCREENING  

Great,  great,  great,  great,  great  

granduncle  was  the  previous  

affected  family  member  

 

~175  years  earlier  

 

Why  should  we  recommend  

expanded  carrier  screening?      

It  has  worked  with  other  ethnic  panels  

 

It  is  recommended  for  the  specialists  

 

It  is  cheaper  that  the  cost  of  maintaining  the  affected  people  

 

Because   populaEon   is   homogenizing   and   ethnicity   panels   are  

not  valid  

 

 

REASONS  TO  DO  IT  

17

Genomic  TesJng  for    

Universal  Carrier  Status    

Test  every  couple  planning  to  conceive  

for  as  many  diseases  as  possible,  

affordably.    

NEW  PARADIGM  

What  diseases    

should  be  included  in    

expanded  screening?    

In   the   NIH   meeEng   on   “PopulaEon-­‐based   Carrier   Screening   for   Single  

Gene  Disorders,”  the  following  criteria  was  recommended:  

§  Impaired  health  in  homozygous  

affected  offspring  

§  High  frequency  of  carriers  in  screened  

populaEon  

§  Technically  and  clinically  valid  

screening  methods  available  

§  Screening  is  cost-­‐effecEve  

§  Consent  is  obtained  

§  IVF,  prenatal  diagnosis  and  terminaEon  

are  reproducEve  opEons  

§  PotenEal  benefits  and  risks  of  carrier  

tesEng  are  explained  pre-­‐  and  post-­‐test  

§  Privacy  is  protected  

§  Experienced  professionals  are  available  

§  SEgmaEzaEon  of  carrier  status  in  the  

community  is  minimized    

The  meeEng  concluded  that  the  top  three  consideraEons  are:  

 carrier  frequency,  disease  burden  and  cost  of  screening.  

1. hkp://www.genome.gov/27026048,  Last  accessed:  9/20/2012    

CRITERIA  FOR  EXPANDED  SCREENING:  NIH  

The  meeEng  concluded  that  the  top  three  consideraEons  are:  

 carrier  frequency,  disease  burden  and  cost  of  screening.  

ACMG & ACOG CURRENT GUIDELINES IN THE UNITED STATES

Cystic Fibrosis ACMG ACOG AJ

Fragile X ACMG ACOG AJ

Spinal Muscular Atrophy ACMG

Hemoglobinopathies ACOG

Bloom Syndrome ACMG AJ

Canavan Disease ACMG ACOG AJ

Familial Dysautonomia ACMG ACOG AJ

Fanconi Anemia Type C ACMG AJ

Gaucher Disease ACMG AJ

Mucolipidosis Type IV ACMG AJ

Niemann-Pick Type A ACMG AJ

Tay-Sachs Disease ACMG ACOG AJ

ACOG = American Congress of

Obstetricians & Gynecologists

ACMG = American College of Medical

Genetics & Genomics

AJ = Ashkenazi Jewish Screening

Recommendations by ACOG/ACMG

Screening  methods:  

SNP  arrays  

Simultaneous analysis of 15,000 of SNPs and/or mutations

Probes for each SNP allele are attached to a slide

DNA sample

SINGLE  NUCLEOTIDE  POLYMORPHISM  (SNP)  MICROARRAYS  

G

A

T

A

A

G

T

A

T

A

A

G

A

G

G

A

T

T

C

T

A

T

T

C

A G

C A A T T A

G

Homozygous  for  C  allele  Heterozygous  C/T  

SNP  MICROARRAYS  

HUMAN GENOME COST Decrease

The  CarrierMap  

•  We   have   created   a   chip   to   determine   carrier   status  

simultaneously   for   more   than   300   geneEc   condiEons   by  

screening  nearly  2,000  mutaEons.    

•  Our  pan-­‐ethnic  screen   includes  diseases  that  are  more  common  

in  specific  ethnic  groups  as  well  as  diseases  that  are  less  common  

or  occur  more  broadly  in  the  general  populaEon.    

 

•  Recombine’s   test   therefore   provides   the   greatest   reducEon   to  

the  overall  risk  of  having  a  child  with  a  geneEc  condiEon.    

THE  RECOMBINE  TEST  

>300 GENETIC DISEASES CARRIERMAP TESTS FOR

Copyright Recombine 2016

Cystic Fibrosis•

Common High Impact Conditions

•

Spinal Muscular Atrophy•

Fragile X Syndrome

Sickle-Cell Disease•

•

Tay-Sachs Disease•

Alpha and Beta Thalassemia

CarrierMap is the most comprehensive genetic carrier screen

available and follows all recommendations and guidelines from the

leading organizations in reproduction and genetics. Useful for all

patients, regardless of ethnicity.

10.05.2016 28

SELECTION CRITERIA DISEASE LIST

10.05.2016 Copyright Recombine 2014 29

INCLUSION: §  Autosomal recessive and/or X-linked recessive inheritance pattern

§  Inclusion in ACOG/ACMG guidelines, and Jewish advocacy group recommendations

§  Inclusion in state newborn screening guidelines

§  Significant impact on life expectancy and/or quality of life, as defined by GC team

§  High carrier rate in at least one population

§  PGD previously performed for genetic disease

§  Meets population-based screening criteria1,2

§  Disease and/or mutation frequency studies published

§  Inclusion in other carrier screening panels

EXCLUSION: §  Autosomal dominant and/or multifactorial inheritance pattern

§  Adult-onset disorder

§  Limited or no impact on reproductive decisions (e.g. MTHFR deficiency)

§  Low penetrance (e.g. HFE-related Hemochromatosis)

1. NIH meeting on “Population-based Carrier Screening for Single Gene Disorders” http://www.genome.gov/27026048, Last accessed: 9/2014.

2. Watson, M.S., Lloyd-Puryear, M.A., Mann, M.Y., Rinaldo, P., Howell, R.R., 2006. Newborn Screening: Toward a Uniform Screening Panel

and System. Genetics in Medicine. 8(5):12S-252S

INCLUSION: §  Autosomal recessive and/or X-linked recessive inheritance pattern

§  Inclusion in ACOG/ACMG guidelines, and Jewish advocacy group recommendations

§  Inclusion in state newborn screening guidelines

§  Significant impact on life expectancy and/or quality of life, as defined by GC team

§  High carrier rate in at least one population

§  PGD previously performed for genetic disease

§  Meets population-based screening criteria1,2

§  Disease and/or mutation frequency studies published

§  Inclusion in other carrier screening panels

EXCLUSION: §  Autosomal dominant and/or multifactorial inheritance pattern

§  Adult-onset disorder

§  Limited or no impact on reproductive decisions (e.g. MTHFR deficiency)

§  Low penetrance (e.g. HFE-related Hemochromatosis)

EVOLUTION CRITERIA DISEASE LIST

10.05.2016 Copyright Recombine 2014 30

NEW DISEASES ADDED BASED ON: §  Emerging literature on natural history and frequency of diseases

§  High carrier frequency in at least one population, particularly in in less well-represented populations (e.g. African, Latin American, Asian, Middle Eastern)

§  Clinical impact affects areas that are less well-studied (e.g. ophthalmology)

NEW MUTATIONS FOR EXISTING DISEASES ADDED BASED ON: §  Emerging literature on natural history and frequency of mutations

§  Associated with diseases for which detection rate is relatively low (<40%)

§  High frequency in at least one population

§  Highly penetrant mutation

DISEASES & MUTATIONS REMOVED BASED ON: §  Emerging literature on relatively low impact of disease and/or mutation

§  Emerging literature on relatively low penetrance of disease and/or mutation

§  Found to be unrelated to reproductive decision-making

HAVE SIGNIFICANT IMPACT RECOMBINE CARRIERMAP DISEASES

10.05.2016 Copyright Recombine 2014 31

185+� Shortened � Lifespan�

α-Thalassemia Cystic Fibrosis

ARPKD

145+� Cognitive � Impairment�

Fragile X Smith-Lemli-Opitz

PKU

290+ � Physical� Impairment�

SCID FMF

β-Thalassemia

Detection Rates

10.05.2016 Genetic Testing. Simplified. 32

Disease Gene

(# Mutations) Ethnic Groups

Detection Rate

Carrier Rate

Residual Risk

Cystic Fibrosis CFTR (99)

African American Ashkenazi Jewish

Asian American European

Hispanic

~81% ~97%

~55% ~93%

~77%

1/61 1/23

1/94 1/25

1/59

1/316 1/767

1/205 1/343

1/257

Spinal Muscular Atrophy SMN1

(Ex7 deletion)

African American Ashkenazi Jewish

Asian American European

Hispanic

~71% ~90%

~93% ~95%

~91%

1/66 1/41

1/53 1/35

1/117

1/121 1/350

1/628 1/632

1/1,061

Bloom Syndrome BLM (2) Ashkenazi Jewish 97% 1/134 1/3,350

Canavan Disease ASPA (4) Ashkenazi Jewish European

99% 47%

1/55 Unknown

1/2,750 Unknown

Familial Dysautonomia IKBKAP (3) Ashkenazi Jewish ~>99% 1/31 <1/3,000

Fanconi Anemia Type C FANCC (6) Ashkenazi Jewish ~99% 1/101 1/10,100

Gaucher Disease GBA (9) Ashkenazi Jewish ~96% 1/15 1/375

Results: Carrier Rates Observed

10.05.2016 33 Copyright Recombine 2013

SAMPLE SIZE & DEMOGRAPHICS

10.05.2016 Copyright Recombine 2014 34

v Sample size: 6,636 samples

§  Analysis limited to samples tested on CarrierMap V3

§  Patient referrals from: endocrinologists, obstetricians, maternal-fetal medicine

specialists, and genetic counselors

§  67% females, 33% males

Females

Males 67%

33%

DEMOGRAPHICS: ETHNICITY

10.05.2016 Copyright Recombine 2014 35

v Patient self-reported ethnicity

§  10 broadly defined ethnic groups, including “Other”

67%

33%

6% 5%

36%

14%

13%

5%

2%

1%

4%

3% 11% African

East Asian

European

Jewish

Latin American

Mediterranean

Middle Eastern

Native American

South Asian

South East Asian

Other

RESULTS: QUICK OVERVIEW

10.05.2016 Copyright Recombine 2014 36

56% of patients identified as carriers

for any disease

39% of patients identified as carriers

for high impact disease

172 diseases, 534 mutations

detected in total

1.9% of couples identified as carriers

for same disease

RESULTS: HIGH IMPACT DISEASES

10.05.2016 Copyright Recombine 2014 37

Disease Counts Percentage

Familial Mediterranean Fever 1/10 10.0%

Spinal Muscular Atrophy 1/17 5.9%

Cystic Fibrosis 1/19 5.2%

Nonsyndromic Hearing Loss & Deafness: GJB2-Related 1/31 3.2%

Alpha Thalassemia 1/51 2.0%

Congenital Disorder of Glycosylation: Type 1A: PMM2 Related 1/51 2.0%

Phenylalanine Hydroxylase Deficiency 1/77 1.3%

Smith-Lemli-Opitz Syndrome 1/77 1.3%

Autosomal Recessive Polycystic Kidney Disease 1/77 1.3%

Beta Thalassemia 1/77 1.3%

10.05.2016 Copyright Recombine 2013 38

Disease Carrier Rate in General Population Observed Reported

Smith-Lemli-Opitz Syndrome 1/47 1/71

GSD Type II: Pompe Disease 1/72 1/97

Nonsyndromic Hearing Loss & Deafness: GJB2 Related 1/19 1/43

Gaucher Disease 1/75 1/112

Bardet-Biedl Syndrome: BBS1 Related 1/168 1/376

Walker-Warburg Syndrome 1/59 1/150

OBSERVED > REPORTED FREQUENCIES

CARRIER OF THE SAME DISEASE

COUPLES

Couples are carriers of the same disease 1,9%�~

*Kumar, N., Bisignano, A., Asgari, S., Chu, B., Munne, S., Grifo, J., Berkeley, A., Licciardi, F., Chen, S., Hoffman, D., Barrionuevo, M., Prates, R.

October 2014. From Carrier Screening to Single Gene PGD: An Analysis of 1/479 Couples Screened via an Expanded Carrier Screening

Platform. ASRM 2014.

29/1479 �

PGD for gene disorders

Disease  tested: Acetil Co Oxidase type I defficiency, Adrenoleucodistrophy, Alpha-thalassemia, Alport syndrome,

Autosomal Dominant Polycystic Kidney Disease (ADPKD), Autosomal Recesive Polycystic Kidney Disease (ARPKD),

Beta-thalassemia, Branchio-Oto-Renal syndrome (BOR), BRCA1 breast cancer predisposition, BRCA2 breast cancer

predisposition, CanavanCharcot-Marie-Tooth type IA (CMT1a), Choroideremia, Congenital adrenal hyperplasia (CAH),

Congenital neutropenia, Connexin 26 hearing loss, Cystic fibrosis, Duchenne/Becker Muscular Dystrophy (DMD),

Ectrodactyly, Ectodermal dysplasia, and Cleft lip/palate syndrome (EEC1), Fabry Disease, Familial adenomatous

poliposis coli (FAP), Familial dysautonomia, Familial intrahepatic cholestasis 2, Fanconi anemia, Fragile site mental

retardation , Gangliosidosis type 1 (GM1), Gaucher disease, Glomuvenous malformations (GVM), Glycogen-storage

disease type I (GSD1), Glycosylation type 1C, Hemoglobin SC disease, Hemophilia A, Hemophilia B, Hereditary

nonpolyposis colon cancer (HNPCC), Hereditary pancreatitis, HLA matching Huntington disease, Hurler syndrome,

Hypophosphatasia, Incontinential pigmenti, Krabbe disease (Globoid cell leukodystrophy), Long QT syndrome, Marfan

syndrome, Meckle gruber, Metachromatic leukodystrophy (MLD), Methylmalonic aciduria cblC type (MMACHC), Myotonic

Dystrophy 1, Myotubular myopathy, Neurofibromatosis 1, Neurofibromatosis 2, Niemann-Pick Disease, Noonan

syndrome, Oculocutaneous albinism 1 (OCA1), Ornithine carbamoyltransferase deficiency (OTC), Osteogenesis

Imperfecta 1, Rapp Hodgkin ectodermal dysplasia, Retinitis pigmentosa, Retinoblastoma, Sickle Cell Anemia, Smith-

Lemli-Opitz syndrome (SLOS), Spinal bulbar muscular atrophy (SBMA), Spinal Muscular Atrophy Type 1 (SMA1), Tay

Sachs, Tuberous sclerosis 1 (TSC1), Tuberous sclerosis 2 (TSC2), Von Hippel-Lindau Syndrome (vHL), X-linked

dominant Charcot–Marie–Tooth (CMTX), etc…… (see review Gutierrez et al. (2008))

   We  can  do  PGD  for  any  of  the  6000  diseases  provided  the  mutaJon  is  known  

DISEASES FOUND IN COUPLES

SUMMARY

High impact disease Carrier

Couples

PGD Cases

Initiated

ALPORT SYNDROME : COL4A4 1 0

CARNITINE PALMITOILTRANSFERASE II 1 0

GLYCOSILATION DEFICIENCY: TYPE 1A, PMM2 1 1

CYSTIC FIBROSIS 6 3

GAUCHER 1 0

MEDITERRANEAN FEVER 2 1

MIOPATHY ON INCLUSION BODIES: TYPE 2 1 1

NIEMANN-PICK : TYPE A 1 1

NON SYNDROMIC DEAFNESS RELATED TO GJB2 7 1

SICKLE CELL DISEASE 4 1

SPINAL MUSCULAR ATROPHY: SMN1 3 2

TAY-SACHS 1 1

Total 29 12

*Kumar, N., Bisignano, A., Asgari, S., Chu, B., Munne, S., Grifo, J., Berkeley, A., Licciardi, F., Chen, S., Hoffman, D., Barrionuevo, M., Prates,

R. October 2014. From Carrier Screening to Single Gene PGD: An Analysis of 1/479 Couples Screened via an Expanded Carrier Screening

Platform. ASRM 2014.

Couple Report

FOR  OOCYTE  DONATION  

OLD  PARADIGM  

•  “we  have  healthy  donors”  

•  Only  few  diseases  

•  Some  characterisEcs  selected  by  phenotype  

 

NEW  PARADIGM  

•  Test  donors  and  couples  for  as  much  diseases  as  possible  

•  Don’t  rule  out  affected  donors  (40%)  but  avoid  matching  (2.4%)  

DONOR  TESTS  

Validation Phase

1 test.

30+ causes of infertility

70+ genes.

700+ variants.

Recombine FertilityMap

The unique test based on the Genetics of Infertility

Designed to provided to patients and

doctors the best options for a success

pregnancy

Validation Phase

Dr. Howard Jones (July 31st 2015)

Thank you Dr. Jones!

Thank you for your attention

jose.horcajadas@gmail.com

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