Genetic Screening of Diseases (It

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Genetic Screening of Diseases (Its worth
potential)

• Dr Derakhshandeh

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First trimester screening for Down syndrome and
trisomy 18

• The availability and acceptability of early
  invasive diagnostic methods (eg, chorionic villus
  sampling)
• The continued need for second trimester screening
  for open fetal neural tube defects

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• Women with singleton pregnancies underwent
  first-trimester combined screening
• measurement of nuchal translucency
• pregnancy-associated plasma protein A PAPP-A
• The free beta subunit of human chorionic
  gonadotropin (HCG) at 10 weeks 3 days through 13
  weeks 6 days of gestation

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Nuchal translucency screening involves the
measurement by ultrasound of the skin thickness
at the back of the neck of a first trimester
fetus
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Nuchal translucency
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Identification about 85-90 of affected fetuses
in the first-trimester

• maternal age was combined with fetal NT
• and maternal serum biochemistry (free ß-hCG and
  pregnancy-associated plasma protein (PAPP-A))

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Second-trimester quadruple screening

• measurement of
• alpha-fetoprotein
• total human chorionic gonadotropin
• unconjugated estriol
• inhibin A at 15 through 18 weeks of gestation

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Maternal blood sampling for fetal blood cells

• This is a new technique
• use of the phenomenon of fetal blood cells
  gaining access to maternal circulation through
  the placental villi
• only a very small number of fetal cells enter the
  maternal circulation in this fashion

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Maternal serum alpha-fetoprotein (MSAFP)

• fetus has two major blood proteins
• albumin and alpha-fetoprotein (AFP)
• Since adults typically have only albumin in their
  blood
• the MSAFP test can be utilized to determine the
  levels of AFP from the fetus

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• the gestational age must be known with certainty
• the amount of MSAFP increasses with gestational
  age
• the MSAFP can be elevated for a variety of
  reasons
• which are not related to fetal neural tube or
  abdominal wall defects, so this test is not 100
  specific

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Neural tube defect

• a neural tube defect in the fetus
• from failure of part of the embryologic neural
  tube to close
• there is a means for escape of more AFP into the
  amniotic fluid

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Neural tube defect
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Prenatal screening and diagnosis of neural tube
defects

• Neural tube defects (NTD) second most prevalent
  congenital anomaly in the United States
• Two factors have played a significant role in the
  prevention of this disorder in developed
  countries
• Sonographic imaging combined with amniocentesis
  for diagnosis of affected fetuses
• folic acid supplements for prevention of the
  disorder

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Anencephaly (failure of closure at the cranial
end of the neural tube)
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Spina bifida (failure of closure at the caudal
end of the neural tube)
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Environmental factors

• The frequency of NTDs is increased with exposure
  to certain environmental factors
• drugs (valproic acid, carbamazepine, Folic acid
  deficiency)
• diabetes mellitus
• Obesity
• Adequate folate is critical for cell division due
  to its essential role in the synthesis of
• nucleic
• certain amino acids

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Genetic factors

• the observations that NTDs have a high rate
• in monozygotic twins
• more frequent among first degree relatives
• more common in females than males
• The risk of recurrence for NTDs approximately 2
  to 4 percent when there is one affected sibling
• With two affected siblings, the risk is
  approximately 10 percent
• The risk of NTD according to family history
• to be higher in countries such as Ireland where
  the prevalence if NTDs is high

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NOTE

• The genetic polymorphisms
• mutations in the methylene tetrahydrofolate
  reductase gene
• may increase the risk for NTDs
• Folate is a cofactor for this enzyme
• which is part of the pathway of homocysteine
  metabolism in cells
• The C677T and the A1298C mutations are associated
  with elevated maternal homocysteine
  concentrations and an increased risk for NTDs in
  fetuses

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Prevention of neural tube defects

• can be accomplished by supplementation of the
  maternal diet with only 4 mg of folic acid per
  day
• but this vitamin supplement must be taken a month
  before conception and through the first trimester

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Maternal serum beta-HCG

• This test is most commonly used as a test for
  pregnancy
• Later in pregnancy in the middle to late second
  trimester
• the beta-HCG can be used in conjunction with the
  MSAFP to screen for chromosomal abnormalities,
  and Down syndrome in particular
• An elevated beta-HCG coupled with a decreased
  MSAFP suggests Down syndrome

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Maternal serum estriol

• made by the fetal adrenal glands
• Estriol tends to be lower when Down syndrome is
  present

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Inhibin-A

• An increased level of inhibin-A is associated
  with an increased risk for trisomy 21
• A high inhibin-A may be associated with a risk
  for preterm delivery

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The facial features of Down syndrome
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overlapping are typical for trisomy 18
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BRCA1 AND BRCA2 GENES
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BRCA1 AND BRCA2 GENES

• Breast cancer develops in about12 percent of
  women who live to age 90
• a positive family history is reported by 15 to 20
  percent of women with breast cancer
• Just only 5 to 6 percent of all breast cancers
  are associated with an inherited gene mutation

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• Two major susceptibility genes for breast cancer,
  BRCA1 and BRCA2
• Testing for mutations in these genes, is
  available
• Clinicians and patients must decide when it is
  appropriate to screen for their presence

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The BRCA1 and BRCA2 genes function

• As an essential part of the normal mechanisms
  that repair double-strand DNA breaks (ionizing
  radiation and DNA cross linking agents such as
  cisplatin)
• through recombination with undamaged, homologous
  DNA strands

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Cisplatin

• Cisplatin is a platinum-based chemotherapy drug
• used to treat various types of cancers, such as
  sarcomas, some carcinomas, lymphomas and germ
  cell tumors

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Cisplatin
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Cisplatin

• works by crosslinking across DNA inter-strands
• making it impossible for rapidly dividing cells
  to duplicate their DNA for cell division
  (mitosis)
• The damaged DNA sets off DNA repair mechanisms
  which fails to work
• so in turn activate cell death processes
  (apoptosis)

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BRCA mutations

• The reason why BRCA mutations predispose mainly
  to breast and ovarian cancers is unclear
• intact BRCA1 represents a barrier to
  transcriptional activation of the estrogen
  receptor
• that functional inactivation could lead to
  altered hormonal regulation of mammary and
  ovarian epithelial proliferation

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BRCA1 and BRCA2 gene abnormalities

• Cancer risk with a high penetrance
• women who have inherited mutations
• the lifetime risk of breast cancer is between 65
  and 85 percent by age 70

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Ovarian cancer

• Ovarian cancer is also linked to the presence of
  BRCA mutations
• the lifetime risk of ovarian cancer
• between 45 and 50 percent in women who have a
  deleterious BRCA1 mutation
• and 15 to 25 percent for those with a BRCA2
  mutation

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BRCA1 and BRCA2-associated cancers

• prostate cancer
• male breast cancer
• pancreatic cancer
• Although the risk of male breast cancer and
  pancreatic cancer may be under 10 percent
• the risk of prostate cancer in BRCA2 carriers may
  be as high as 35 to 40 percent

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BRCA 1

• The gene Locus for BRCA1 17q21
• a large gene
• 24 exons
• encoding a 220 kD
• 1863 amino acids
• Two recognizable motifs

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BRCA2

• BRCA2 (13q12.3)
• was identified by Wooster et al. in 1995
• It encodes for 384 kD nuclear protein
• 3418 amino acids
• BRCA2 bears no homology to any known tumour
  supressor genes
• contains 27 exons
• spread over 70 kb of genomic DNA

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Breast Cancer Families
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Significance of family history

• Degree of relatedness to affected relatives
• Number of affected relatives
• The age of the relative(s) when breast cancer
  occurred
• Laterality of the disease in affected relatives
• Whether there is a family history of ovarian
  cancer

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Pedigree of Breast cancer FamilyWith BRCA1
mutation
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SSCP AnalysisExon 11pi BRCA1 MS R1347G
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Mutations in BRCA1/2 gene
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• Screening for colorectal cancer

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Screening for colorectal cancer

• Colorectal cancer (CRC) is
• a common
• Lethal
• preventable disease
• It is infrequent before age 40
• the incidence rises progressively to 3.7/1000 per
  year by age 80

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Clinical detection of increased risk

• Before deciding how to screen
• clinicians should decide whether the individual
  patient is at average or increased risk
• based on his or her medical and family history
• A few simple questions are all that is necessary
• Have you ever had colorectal cancer
• or an adenomatous polyp
• Have you had inflammatory bowel disease (Crohn
  disease)
• Has a family member had colorectal cancer or an
  adenomatous polyp
• If so, how many
• was it a first-degree relative (parent, sibling,
  or child)
• and at what age was the cancer or polyp first
  diagnosed

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Crohns disease

• an inflammatory bowel disease
• causes inflammation of the gastrointestinal tract
  in both men and women
• persistent diarrhea, abdominal pain, fever, and
  at times rectal bleeding

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Crohns disease
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screening for Colorectal cancer (CRC)

• Patients at highest risk with familial syndromes
  (HNPCC, FAP)
• should be screened for CRC with colonoscopy at
  frequent specified intervals

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People at high risk

• a first-degree relative with colon cancer
• or adenomatous polyp diagnosed at age lt60 years
• or two first-degree relatives diagnosed at any
  age
• should be advised to have screening colonoscopy
  starting at age 40 years
• or 10 years younger than the earliest diagnosis
  in their family
• whichever comes first, and repeated every five
  years

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Adenomatous polyp
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Microsatellite Instability in Adenomas as a
Marker for Hereditary Nonpolyposis Colorectal
Cancer

• Hereditary nonpolyposis colorectal cancer (HNPCC)
• the most common of the well-defined colorectal
  cancer syndromes
• accounting for at least 2 of the total
  colorectal cancer
• carrying a greater than 80 lifetime risk of
  cancer

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Microsatellite instability (MSI)

• can be detected in approximately 90 of tumors
  from individuals with Hereditary Non-Polyposis
  Colorectal Cancer (HNPCC)
• MSI is also reported in approximately 15 of
  sporadic colorectal carcinomas

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Mutations in the human mismatch repair genes
(MLH1, MSH2, PMS1, PMS2, MSH6)

• responsible for the MSI of the HNPCC tumors
• In contrast to the classical tumor suppressor
  pathway
• the mismatch-repair gene tumor pathway
  accumulates mutations in genes involved in
  tumorigenesis

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Reduction in cancer morbidity and mortality of
HNPCC patients

• can be accomplished by appropriate clinical
  cancer screening of HNPCC patients with mutations
  in mismatch repair (MMR) genes

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Germline mutation analysis

• In individuals with cancer
• mutation detection can be accomplished relatively
  efficiently by germline mutation analysis of
  individuals (blood) whose cancers show
  microsatellite instability (MSI)

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• Among 378 adenoma patients
• six (1.6) had at least one MSI adenoma
• Five out of the six patients (83) had a germline
  MMR gene mutation
• MSI analysis is a useful method of prescreening
  colorectal adenoma patients for HNPCC
• Microsatellite Instability in Adenomas as a
  Marker for Hereditary Nonpolyposis Colorectal
  Cancer
• Anu Loukola et al. American Journal of Pathology.
  19991551849-1853

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Somatic mutations

• Cells deficient for both alleles of a mismatch
  repair gene, leading to somatic mutations
• which can be demonstrated by analyzing
  microsatellite sequences in the tumor DNA
• These sequences display frequent somatic
  deletions and insertions, often referred to as
  microsatellite instability (MSI).
• HNPCC patients form adenomas at a slightly but
  not strikingly increased rate as compared with
  the general population

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The frequency of the MSI

• The frequency of the MSI is 80 to 95 in HNPCC
  cancers
• 10 to 15 in sporadic colorectal cancers

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Factor V Leiden Assay on LightCycler

• The presence of the factor V mutation can cause
  an increased risk of venous thrombosis
• Individuals heterozygous for the Factor V Leiden
  (FacV) mutation
• carry an eight-fold greater risk for thrombosis
• while homozygosity confers an estimated
  ninety-fold increased risk

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SMA Carrier Testing
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Screening for cardiovascular risk with C-reactive
protein
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Screening for cardiovascular risk with C-reactive
protein

• The most extensively studied biomarker of
  inflammation in cardiovascular diseases
• serum C-reactive protein (CRP)

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• Elevated serum CRP was a strong independent risk
  factor for cardiovascular disease
• added to the predictive value of other factors,
  such as serum total cholesterol
• Elevated serum CRP was a stronger predictor of
  cardiovascular events than LDL cholesterol
  (LDL-C) At eight years

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Screening for coronary heart disease

• Although mortality from coronary heart disease
  (CHD) has fallen substantially over the past
  three decades
• it remains the leading cause of death in adults

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High-risk patients

• risk factors for CHD, including
• Hypertension
• Hypercholesterolemia
• Smoking
• a family history of premature CHD
• and diabetes mellitus

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• The Association Between Apolipoprotein E
• Polymorphism and Cardiovascular Risk Factors

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Association Between Apolipoprotein EPolymorphism
and Cardiovascular Risk Factors

• the allelic and genotypic frequencies related to
  apolipoprotein E (ApoE) polymorphism
• association of the genotypes with risk factors
• and cardiovascular morbidity in population

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• the gene amplification technique through the
  polymerase chain reaction-restriction fragment
  length polymorphism
• (PCR-RFLP) and cleavage with the restriction
• enzyme Hha I to identify the ApoE genotypes The
  most frequent
• Individuals with the E3E4 had a mean age greater
  than those with the E3E3

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• Cystic fibrosis Prenatal genetic screening

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Cystic fibrosis Prenatal genetic screening

• Cystic fibrosis (CF) is a chronic pulmonary
• and exocrine pancreatic disease
• the most common monogenic disorder in Caucasians
  of Northern European
• classic form it is marked by abnormal sweat
  chloride levels
• chronic pulmonary disease
• pancreatic insufficiency
• liver disease
• and obstructive azoospermia in males

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Cystic fibrosis

• an autosomal recessive disease
• with a carrier rate of 1 in 22 to 25 in Caucasian
  Americans of Northern European background
• the most common mutation in this group is called
  F508 and accounts for 75 percent of all CF cases

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The effect of ethnicity on CF screening

• The key to all CF screening is knowing which
  mutations to test for
• knowledge of the most common CF mutations in
  individuals of different heritages
• Native Americans are at increased risk of CF
  (carrier frequency 1 in 31
• Jewish heritage (carrier rates ranging from 1 in
  24 to 1 in 29 if the patient's ancestors
  originated in Greece, Bulgaria, or Libya)
• to 1 in 90 if the ancestors originated in Iran or
  Iraq)

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PHENOTYPE of CF

• The diagnosis and treatment is complicated
• not everyone who is homozygous for the CF
  mutation has the classic form of the disease
• Some individuals have an atypical presentation
• pulmonary disease associated with pancreatic
  sufficiency
• have only isolated features of the disease
• pancreatitis
• liver disease
• nasal polyps
• congenital bilateral absence of the vas deferens
  (CBAVD)

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Vas deferens (CBAVD)
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Prenatal testing for the hemoglobinopathies and
thalassemias

• hemoglobinopathies as two general types
• the thalassemias
• decreased globin chain production
• hemoglobin variants (eg, sickle cell anemia and
  its variants, hemoglobin C disease)
• chronic, debilitating, and often fatal
• new therapies
• including hydroxyurea (XMNI, g mRNAgtactive)
• hematopoietic cell transplantation
• gene therapy

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Identification of candidates for prenatal testing

• Family history of a relative with a
  hemoglobinopathy or thalassemia
• couples from extended families living in endemic
  areas
• consanguineous marriages are common, may be at
  highest risk
• Ethnic background
• at low risk for hemoglobinopathies are northern
  Europeans
• Japanese
• Native Americans

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Red blood cell indices

• mean corpuscular volume (MCV) lt80 femtoliters
  (fL), MCHlt 28
• in the absence of iron deficiency (alpha or beta
  thalassemia)
• Hemoglobin electrophoresis
• this test will identify carriers
• hemoglobin variants
• beta thalassemia