This document discusses complex or multifactorial diseases. It provides examples such as various congenital malformations, non-communicable diseases, and certain cancers. It explains that these diseases are caused by multiple contributing genetic and environmental factors, rather than a single gene. They do not follow clear Mendelian patterns of inheritance and can be influenced by environmental exposures. The document then discusses thrombophilia and inherited breast cancer as examples, outlining various genetic and non-genetic risk factors involved in the development and progression of these complex diseases.

1. Complex (multifactorial) diseases
Ekbal Mohamed Abo-Hashem-MD
Professor of Clinical Pathology
Mansoura University-Egypt .

2.  Conditions caused by many contributing factors are called
complex or multifactorial disorders.
 Although complex disorders often cluster in families, they
do not have a clear-cut pattern of inheritance. This makes
it difficult to determine a person’s risk of inheriting or
passing on these disorders.
 Complex disorders are also difficult to study and treat
because the specific factors that cause most of these
disorders have not yet been identified.

3. What Are the Characteristics of a Multifactorial Disease?
A multifactorial disease has a combination of distinctive
characteristics that can be differentiated from clear-cut Mendelian
or sex-linked conditions. These traits include the following:
•The disease can occur in isolation, with affected children born to
unaffected parents. Although familial aggregation is also common
(i.e., there may be multiple cases in the same family), there is no
clear Mendelian pattern of inheritance.
•Environmental influences can increase or decrease the risk of the
disease.

4. •The disease occurs more frequently in one gender than in the other,
but it is not a sex-limited trait. In addition, first-degree relatives of
individuals belonging to the more rarely affected gender have a higher
risk of bearing the disease
•The concordance rates in monozygotic and dizygotic twins contradict
Mendelian proportions. A concordance rate is a measure of the rate at
which both twins bear a specific disease.
• The disease occurs more frequently in a specific ethnic group (i.e.,
Caucasians, Africans, Asians, Hispanics, etc.).
(cont.)

5.  Examples of complex disorders :
*Congenital malformations
congenital heart diseases
Neural tube defects
Cleft lip/palate
*Non-communicable diseases :
Diabetes mellitus
Hypertension
Obesity
 Hereditary breast and colon cancer
Asthma
Alzheimer disease
schizophrenia
Thrombocytosis
Pyloric stenosis
Congenital hip dysplasia

6. For diseases caused by alterations in just one gene, such as
cystic fibrosis, Huntington's disease, or Marfan syndrome, the
journey starts by finding the gene; determining the protein
the gene instructs the cell to make; and learning the role that
protein plays in cell function. Translating this information into
treatments or cures, is the key aim for genetic research .

7. In the so-called diseases of civilization - complex disorders,
such as high blood pressure and other familiar diseases of the
heart and circulatory system, diabetes, obesity, cancer,
psychiatric illness, asthma, arthritis - pose difficult health
problems because there is still little information about what
causes them and even less about how to prevent or cure
them.

8. 1. Thrombophilia

10. Risk Factors :

11. a. Genes - Environment Interaction
Venous thrombosis is caused by a disruption of normal hemostasis caused by the
interaction of one or more genes and environmental factors and/or acquired condi-
tions, including use of oral contraceptives, trauma, obesity, immobility, pregnancy,
age, or surgery. Although the protein products of many genes are involved in the
anticoagulation and coagulation pathway and are involved in the regulation of
hemostasis, mutations in the factor V and prothrombin genes are associated with
DVT, represent the most frequent genetic cause of thrombophilia, and are common
in the population

12. Activated protein C (APC) in conjunction with its cofactor, protein S,
plays a key role in the anticoagulant system by inactivating membrane-
bound factors Va and VIIIa. The inability to inactivate procoagulant
factors Va or VIIIa could disturb hemostasis, heighten the coagulation
pathway, increase the generation of thrombin, and promote clot for-
mation.

13. b. Factor V gene and APC
A linkage of the factor V gene and the APC resistance phenotype was
observed and reported a G-to-A base substitution at nucleotide 1691 in exon
10 of the factor V gene. This nucleotide change results in an arginine-to-
glutamine substitution at codon 506 (R506Q) in the factor V protein.
Cleavage at the arginine residue at codon 506 by APC is the initial step of
inactivation of the activated factor V protein, causing it to display a
decreased affinity for factor Xa. As a result, there is reduced efficiency in
catalyzing the activation of prothrombin to alpha-thrombin..

14. However, substitution with a glutamine residue at this site prolongs
inactivation of this molecule by APC by approximately tenfold, thereby
shifting the balance of hemostasis to favor coagulation and increasing
thrombin production. Heterozygous carriers have a 7.9-fold increased
relative risk of thrombosis compared with a ninety-one fold increased risk for
homozygotes.

15. c. G 20210A
A genetic variant in the 3'-untranslated region of the prothrombin gene
was described in patients with a documented family history of venous
thrombosis. This common allele (G-to-A base substitution at nucleotide
20210) increased the risk of venous thrombosis and was associated with
increased plasma prothrombin (> 1.15kU/L). The risk of venous
thrombosis is increased sixteen fold in G20210A carriers using oral
contraceptives, and the risk of cerebral vein thrombosis increases 149-
fold in G20210A carriers using oral contraceptives.

16. Prothrombin, also referred to as factor II, is the precursor of thrombin
and plays a primary role in fibrin production and clot formation. The G-
to-A substitution increases the processing of the 3' -end of the pre-
mRNA and thus functions as a gain-of-function mutation, culminating in
increased mRNA accumulation and increased synthesis of protein. This
aberration of RNA metabolism results in increased synthesis of pro-
thrombin and can enhance clot formation. Note that the substitution
does not change the amino acid sequence of prothrombin.

17. Inherited together, G1691A (factor V) and G20210A (factor II,
prothrombin) convey at least a twenty fold increased risk for a
venous thromboembolic event (VTE). They are commonly seen
together in thrombophilia patients thus supporting the additive
genetic effect associated with complex diseases.

19. d. DNA Testing
DNA testing for G1691A and G20210A in clinical laboratories is
performed by anyone of several methodologies with those most
commonly reported being invasive cleavage of oligonucleotide probes
(Invader assay), PCR coupled with restriction-endonuclease digestion
and gel electrophoresis, and real-time PCR. Many other techniques
have been described.

20. Benefits from DNA testing include the identification of individuals at risk
for recurrent events, especially in situations that predispose to
thrombosis, such as oral contraceptive use, management of pregnancy
complications, or hormone replacement therapy. In addition, DNA testing
enables the identification of at-risk family members

21. 2. Inherited Breast Cancer

24. Mutations in the two major breast cancer genes,
BRCA 1 and BRCA2, predispose patients to breast
and ovarian cancer and to prostate and colon cancer
(BRCA1) or pancreatic cancer (BRCA2).
a. BRCA1 and BRCA2 Mutation Predisposes to and Accelerates the Progression of Caner

29. The progression rate of breast neoplasia is accelerated in women who carry
BRCA1 or BRCA2 mutations compared with other patients who have breast
carcinoma with or without a family history.
In families in which breast cancer is segregating, but no BRCA1 or BRCA2
mutation has been detected, additional genes that predispose to breast cancer
are likely but have yet to be identified.
a. BRCA1 and BRCA2 Mutation Predisposes to and Accelerates the Progression of Caner
(cont.)

30. The inability to identify breast cancer susceptibility genes in these families may reflect:
(1) genetic heterogeneity in the family with mutations in several genes,
(2) low penetrance of these mutations, making it difficult to distinguish family members
without mutation from asymptomatic carriers in the studies,
(3) an autosomal recessive mode of inheritance, or
(4) breast cancer acting as a complex disease that results from the interaction of both
several genes and environmental factors, thereby making it difficult to tease out the genetic
component of the disease.
b. Causes of Absence of Susceptibility Genes Detection

31. Recently developed gene-expression assays of tumor tissue may be instrumental
for classification of these families into subsets, aiding studies aimed at deter-
mining the molecular origin of these cancers. Mutations in tumor-suppressor
genes TP53 or ATM are also associated with "familial" cancer, including breast
cancer in mutation positive females.

32. Mutations in BRCA1 and BRCA2 are inherited in an autosomal dominant fashion,
with offspring of known carriers or of affected patients possessing a 50% chance
of inheriting the predisposing cancer gene mutation.
Inheritance of the mutation does not convey a certainty of developing cancer
nor indicate the type of cancer or the age of onset.
c. Mode of Inheritance and Cumulative Risk

33. The average cumulative risk of breast cancer for mutations in either BRCAI or
BRCA2 is about 27% to age 50 and 64% to age 70.
Both environmental and genetic factors play role in the development of breast
or other cancers in mutation-positive patients as does the type of DNA mutation
in BRCA1 or BRCA2.

34. Using DNA linkage studies, the gene was mapped for early onset familial breast
cancer to 17 q21 (long arm of chromosome 17 banding region 21).
BRCA1 was cloned and later confirmed as the susceptibility gene in breast and
ovarian cancer kindreds.
d. BRCA1 and BRCA2 Tumor-Suppressor Genes

36. The BRCA2 gene, unrelated in sequence to BRCA1, spans 70kb, contains 26
exons, encodes an 11.5-kb mRNA, and is translated into a protein of 3418 amino
acids.
BRCA1 and BRCA2 are considered tumor-suppressor genes requiring inactivation
of both alleles for progression to neoplasm.

37. In a patient with familial breast cancer, a mutant allele is inherited, and the
second allele-the patient's wild-type allele-is inactivated through somatic
mutation.
BRCAI and BRCA2 proteins are multifunctional, interacting with numerous other
proteins in complex and separate systems involved in response to DNA damage,
regulation of transcription, remodeling of chromatin, and regulation of cell
growth.

38. Testing for disease-associated mutations is made difficult by the heterogeneity
of disease-causing mutations and the complexity of the BRCA1 and BRCA2 genes.
e. Heterogeneity of Mutation

39. Interestingly the majority of BRCAI and BRCA2 disease-associated mutations
result in premature truncation of the protein and thus a loss of function.
For this reason, the protein truncation test (PTT) is often employed as a
screening method for mutation detection.
f. Protein Premature Truncation and Protein Truncation Test

40. In this methodology, multiple PCR primer pairs are designed to span the gene
with each primer pair, including one primer that includes an RNA polymerase
promoter and a translation initiation sequence.
In-vitro translation system, and the synthesized proteins are subjected to
sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis for detection of
truncated proteins.
(Cont.)

41. Common screening assays include microarrays, denaturing high-performance
liquid chromatography (DHPLC), single-strand conformation polymorphism
(SSCP), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis
(HA), and fluorescence assisted mismatch analysis (FAMA).
g. DNA Screening Assays

42. More recently, other unique methods have been described as well, including
allele-specific gene expression analysis (AGE), multiplex ligation-dependent
probe amplification (MLPA), and restriction endonuclease fingerprinting, single-
strand conformation polymorphism coupled with capillary electrophoresis.

43. Although screening assays can detect DNA perturbations, DNA sequence (DS)
analysis is required for precise identification of the base or bases involved.
h. Importance of DNA Sequencing

44. In many instances, a combination of screening methods is used, thereby
reducing the region of the gene that requires DNA sequencing analysis.
An alternative approach is to eliminate screening of the gene and rather
perform direct DS analysis of the BRCA1 and BRCA2 genes on each specimen.

45. BRCA1 or BRCA2 mutation in a patient is increased to 10% or greater if:
 Breast cancer was diagnosed in two women in the family before the age of 50.
 Breast cancer was diagnosed in women in the family before age 50, and ovarian cancer
was detected in one or more women in the family.
The likelihood of mutation

46. 3. Inherited Colon Cancer

49. Molecular pathways of colon cancer

50. The molecular basis of sporadic and inherited CRC involves two distinct
pathways :
- Chromosomal instability pathway
- Microsatellite instability pathway
The original model of chromosome instability has been further characterized to
reveal a complex chain of events whereby normal colon lining (mucosa) is
transformed into adenomatous and then into malignant mucosa via the
inactivation of tumor-suppressor genes and the activation of genes involved in
tumor cell proliferation.

52. The chromosomal instability (CIN) pathway begins with the loss of function of
the adenomatous polyposis coli (APC) tumor-suppressor gene product, most
often because of a somatic inactivating gene mutation on one allele followed by
a chromosomal deletion encompassing the second APC allele and adjacent
flanking DNA on chromosome 5q.
Since APC is involved early in the tumorigenic process, it has been referred to as
the "gatekeeper".
a. Chromosomal Instability Pathway

53. The cascade of events proceeds with continued activation of the KRAS (Kirsten
rat sarcoma virus) protooncogene on 12p 12.1 (short arm of chromosome 12
banding region 12.1) through somatic gene mutations (most frequently occurring
in codons 12, 13, or 61), which in the presence of APC inactivation increases
growth and proliferation of the cell.

54. Subsequent inactivation of the tumor-suppressor gene DCC (deleted in colon
cancer) and frequent loss of adjacent tumor-suppressor genes on 18q (long arm
of chromosome 18) including SMAD4 and SMAD and the inactivation of tumor-
suppressor gene TP53 on 17p (short arm of chromosome 17) are identified in late
adenoma and carcinoma.

56. The microsatellite instability (MSI) pathway in sporadic CRC arises from
mutations or altered expression of genes involved in DNA mismatch repair
(MMR). As a result of an altered and thus dysfunctional MMR system, DNA
replication errors, primarily within microsatellite repeats or repetitive sequences,
remain uncorrected and accumulate
b. microsatellite instability pathway

57. Inherited CRC syndromes initiate as a result of an inherited mutation in one of
the genes involved in the CIN or MSI pathway. Although several CRC syndromes
exist, the two most common are familial adenomatous polyposis (FAP) and
hereditary nonpolyposis colorectal cancer (HNPCC).
c. Familial Adenomatous Polyposis and Hereditary Non-plyposis
Colorectal Cancer

58. The gene responsible for FAP, the APC gene, was cloned following linkage to
chromosome 5q21 (long arm of chromosome band 21) in FAP kindreds.
d. APC Gene and Protein

59. The APC gene spans 8535 base pairs, contains 15 exons, and encodes a protein
of 2843 amino acids with a molecular weight of about 312kD, The APC protein is
a multidomain, multifunctional protein, participating in several cellular
processes, including cell adhesion and migration, signal transduction,
microtubule assembly, and chromosome segregation.
Best understood is the regulation of -catenin levels through interaction with
APC.

60. -catenin is required for binding with E-cadherin, a member of the calcium-
dependent cell adhesion molecules, and is also involved in signal transduction
pathways. Mutations altering the association of -catenin, with APC minimize
degradation and increase cytoplasmic levels of -catenin; this can affect cell-cell
adhesion and the transcription of genes, promoting cell proliferation (specifically
CMYC) or inhibition of cell death.

61. Studies on FAP families indicate that a wide variety of germline mutations exist;
>95% result in truncated proteins, either because of a nonsense mutation (30%)
or by a frameshift mutation, and most are contained within the 5`-half of the
gene.
e. Germline Mutation and Truncated Protein
In germline mutations there are two hot spots at codons 1061 and 1309, with
the most common mutation being an AAAAG deletion at codon 1309.
These mutations leave the truncated APC protein unable to regulate -catenin.

62. Genotype-phenotype correlations exist for some APC mutations. Patients with
truncating mutations at the extreme 5`-end of the gene (codons 1 to 163) or
mutations at the carboxyl-terminal end of the gene (codons 1860 to 1987) have
the attenuated form of the disease, developing a smaller number of polyps
(<100).
f. Genotype-phenotype correlations
A severe phenotype is observed in patients with mutations between codons 1250
and 1464, and patients with truncating mutations between codons 1403 and 1578
are at an increased risk for extracolonic disease.

63. The protein truncation test (PTT) is most often used to test for mutations in a
family because the majority of FAP mutations result in a truncated protein. Once
a possible mutation has been detected, the region is sequenced to determine the
precise identity of the mutation.
g. DNA Testing

64. Other screening or direct detection methodologies can be used, and collectively
a sensitivity of about 87% can be achieved. If the mutation within the family can
be identified, it is recommended that the family be referred for genetic
counseling.
DNA testing should be performed in at-risk family members as young as 10 to 12
years of age. Genetic testing can significantly alter the 50% pretest risk for
disease to a risk of 0% or 100%.
(cont,)

66. The most common inherited CRC susceptibility syndrome is HNPCC, which
represents about 2% to 3% of all CRC cases. In contrast to FAP, HNPCC is
characterized by a few polyps that possess an accelerated transformation poten-
tial to carcinoma in as little as 1 to 2 years.
HNPCC is inherited as an autosomal dominant disorder with a penetrance of
80% to 85%. HNPCC patients have a lifetime risk of 70% to 80% for developing
CRC, thereby suggesting a role for other factors in this disease process.
h. HNPCC

67. Mutations in six mismatch repair genes have now been linked to HNPCC. The
first was mapped to 2p 15-16.
Simultaneously, MSI was noted in a subset of sporadic CRC. One such gene,
hMSH2, mapped to 2p15-16 and in fact was found to be altered in HNPCC
kindreds.
i. Genes Linked to HNPCC
Subsequently, mutations in genes hMSH6 (2pI5-16), hMLHI (3p21), hPMSI
(2q3l), hPMS2 (7p22), and hMLH3 (14q24.3) have been identified in HNPCC
families, although more than 90% of HNPCC mutations are observed in
hMSH2 and hMLHI.

68. • HNPCC mutations are diverse and are located throughout these genes. Almost
all errors made during DNA replication are repaired through the proofreading
3'-to-5' exonuclease activity of DNA polymerase.
• Uncorrected errors of mismatched bases between the two strands are
repaired before cell division through the MMR proteins
j. HNPCC and Errors in Mismatch Repair

69. • Testing of tumor tissue for MSI is the initial laboratory step in investigation of
HNPCC patients because MSI is a measure of MMR deficiency and indicates
probable defects in MMR genes through germline and somatic changes,
International guidelines for analysis of MSI in CRC recommend a panel of five
markers: BAT25, BAT26, D5S346, D2S123, and D 17S250. MSI is characterized
by the expansion or contraction of DNA sequences through the insertion or
deletion of repeated sequences.
k. MSI Testing, Sequence Analysis and PTT