A/34-38 GENETIC DISORDERS AND DEVELOPMENTAL ABNORMALITIES (Leiel)

Question 1

Which factors have enabled the rapid expansion of molecular diagnostics?

Answer 1

1. the sequencing of the human genome and the deposition of these data in publicly available databases.
2. the availability of numerous “off-the-shelf” polymerase chain reaction (PCR) kits tailor-made for the identification of specific genetic disorders.
3. the availability of high-resolution microarrays (“gene chips”) that can interrogate both DNA and RNA on a genomewide scale using a single platform.
4. the emergence of automated, high throughput, next-generation (“NextGen”) sequencing technologies.

Question 2

When assessing a genetic aberration what should be considered in terms of sampling?

Answer 2

the genetic aberration being queried can be either in the:

germline (i.e., present in each and every cell of the affected person, as with a CFTR mutation in a patient with CF)

or

somatic (i.e., restricted to specific tissue types or lesions, as with MYCN amplification in neuroblastoma cells).

This consideration determines the nature of the sample (e.g., peripheral blood lymphocytes [PBLs], saliva, tumor tissue) used for the assay.

Question 3

The indications for genetic analysis can be divided

into:

Answer 3

• Inherited conditions
  
  • Genetic testing can be offered at either the prenatal or postnatal stages.
• Acquired conditions

Question 4

What are the Genetic Analysis techniques?

Answer 4

It may involve:

• conventional cytogenetics (karyotyping)
• FISH (Fluorescent in situ hybridization)
• molecular diagnostics (collection of techniques used to analyse biological markers in the genome)
• combination of these techniques.

Question 5

Prenatal genetic analysis should be offered to

Answer 5

All patients who are at risk of having cytogenetically abnormal progeny.

Question 6

On which cells can it be preformed?

Answer 6

It can be performed on cells obtained by amniocentesis (sampling of amniotic fluid), on chorionic villus biopsy material, or increasingly in “liquid biopsies” on maternal blood paired with next-generation sequencing.

Question 7

Waht are some indications for prenatal genetic analysis?

Answer 7

Some important indications are the following:

• Advanced maternal age (beyond 34 years), which is associated with greater risk of trisomies.
• Confirmed carrier status for a:
  
  • balanced reciprocal translocation
  • robertsonian translocation
  • inversion
  • (in such cases, the gametes may be unbalanced, so the progeny would be at risk for chromosomal disorders)
• Fetal abnormalities observed on ultrasound, or an abnormal result on routine maternal blood screening
• A chromosomal abnormality or mendelian disorder affecting a previous child
• Determination of fetal sex when the patient or partner is a confirmed carrier of an X-linked genetic disorder.

Question 8

On which cell does postnatal genetic analysis is usually preformed?

What are the indications for postnatal genetic analysis?

Answer 8

Postnatal genetic analysis usually is performed on peripheral blood lymphocytes. Indications are as follows:

• Multiple congenital anomalies
• Suspicion of a metabolic syndrome
• Unexplained mental retardation and/or developmental delay
• Suspected aneuploidy (e.g., features of Down syndrome) or other syndromic chromosomal abnormality (e.g., deletions, inversions)
• Suspected monogenic disease, whether previously described or unknown

Question 9

Acquired genetic alterations

Answer 9

Acquired genetic alterations, such as somatic mutations in cancer, are increasingly becoming a large focus area in molecular diagnostics laboratories, especially with the advent of targeted therapies. Although single gene tests (mutations of EGFR or BRAF, amplification of HER2) have been used for a while to inform treatment decisions, the advent of cost-effective next-generation sequencing approaches has now allowed interrogations of large numbers of coding genes (often in the 100s), as well as cancer-relevant translocations, in a single assay.

The clinical team typically receives a “genomic report” on the patient’s cancer, including potential molecularly targeted treatment recommendations.

Another major focus of molecular diagnostics has been the rapid identification of infectious diseases (such as suspected tuberculosis or virulent pathogens such as Ebola) using DNA-based approaches.

In general, these approaches have cut down the time required for diagnosis from weeks to a matter of days. Besides de novo identification of pathogens, molecular diagnostics laboratories can also contribute to the identification of treatment resistance (e.g., acquired mutations in influenza viruses that render them resistant to anti-virals), and to the monitoring of treatment efficacy using assays for “viral load” in the blood. Similar parameters (measuring efficacy of therapy and emergence of resistance) are also widely used in cancer patients.

Because of the rapid advances in molecular diagnostics, terms such as “personalized therapy” and “precision medicine” are being increasingly used to indicate therapy tailored to the needs of the individual patient.

Question 10

Which analyitic test is used for the detection of chromosomal copy number abnormalities? (wht is it’s disadvantage?)

At which levels can chromosomal copy number abnormalities occur?

How are subchromosomal alterations identified?

Answer 10

Karyotype analysis of chromosomes by G banding remains the classic approach for identifying changes at the chromosomal level (the resolution with this technique is fairly low)

• at the level of the entire chromosome (trisomy 21)
• chromosomal segments (22q11 deletion syndrome)
• submicroscopic intragenic deletions (WAGR syndrome).

To identify subchromosomal alterations, both focused analysis of chromosomal regions by FISH and global genomic approaches such as comparative genomic hybridization (CGH).

Question 11

What are the patterns of inheritance of Mutations involving one gene?

Answer 11

• Autosomal dominant
• Autosomal recessive
• X-linked.

Single-gene defects follow the mendelian pattern of inheritance.

Question 12

List some characteristics of autosomal dominant inheritance

Answer 12

• Manifested in the heterozygous
• At least one parent of the index case if affected. Men and women are equally affected.
• Both sexes can transmit the disease
• Probability to inherit the disease Is 50% if one parent is affected
• Some patient gain the disease due to a new mutation (that is, they do not have affected parents)
• Features can be modifies by reduced penetrance (the mutant gene is present but the person is phenotypically normal) or by variable expressivity (same mutant gene is expressed differently among different individuals).
• onset might be in adulthood (Huntington disease)
• “dominant negative protein”=the product of a mutant allele that inhibits the function of the product of normal allele.
• enzyme proteins are not affected. In autosomal dominant disorders, the affected protein is:
  
  • Receptor protein (LDL receptor in familial hypercholesterinaemia)
  • Structural protein (collagen)

Question 13

List some common autosomal dominant disorders:

Answer 13

• Nervous
  
  • Huntington, neurofibromatosis, myotonic dystrophy
• Urinary
  
  • Polycystic kidney disease- APC gene mutation
• GI
  
  • Familial polyposis coli
• Hematopoietic
  
  • Von Willebrand disease
  • Hereditary spherocytosis
• Skeletal
  
  • Marfan syndrome- fibrillin mutation
  • Ehlers-Danlos: defect of collagen synthesis (6 variant)
• Metabolic
  
  • Familial hypercholeteremia
  • Acute intermittent porphyria

Question 14

What is the pathogenesis of Marfan syndrome?

Answer 14

• Mutated gene: FBN1, codes for a glycoprotein called fibrillin 1.
• Function of fibrillin 1: a component of microfibrils, which are component of elastic fibers → connective tissues are affected.
• Probably, another mutation in involved in the disease: increased production of TGF-β (transforming growth factor beta). This cytokine is responsible for the regulation of connective tissue growth and architecture → overgrowth of bones and changes in the mitral valve.

Question 15

What is the morphology and clinical manifestation of Marfan syndrome?

Answer 15

Morphology:

• skeletal abnormalities
  
  • Slender elongated habitus, abnormally long legs, arms and fingers. Arched-palate, hyperextensibility of joints. Spinal deformities, depressed sternum.
• eyes
  
  • Bilateral dislocation of the lens due to weakness of suspensory ligament
• cardiovascular system
  
  • Aneurysm in the aorta, aortic dissection, aortic valve incompetence (due to dilation)
  • Cardiac valves, especially the mitral, become excessively distensible → regurgitation → congestive heart failure

Clinical manifestation

• Clinical expression is variable
• Most common cause of death is from aortic rupture. Less commonly it is cardiac failure.

Question 16

Describe the pathogenesis and the molecular basis of Danlos-Ehlers syndromes.

Answer 16

Pathogenesis

• Defect in the collagen synthesis or structure.
  
  • (There are approximately 30 types of collagen, each In the product of different gene. )
• There are 6 clinical and genetic variants of EDS.

Molecular basis of EDS:

1. Mutation of COL3A1 → deficiency of collagen type 3
2. Mutation of COL1A1 and COL1A2 → mutation of type 1 collagen
3. Deficiency of enzyme lysyl hydroxylase → defect in the crosslinks among collagen

Question 17

Describe the clinical picture associated with Danlos-Ehlers syndromes.

Answer 17

Skin:

• Hyper-extensible, extremely fragile → vulnarable to trauma.

Joints:

• Hyper-mobile → vulnarable to dislocation.

Complication in internal organs:

• Rupture of the colon, large arteries, cornea.
• Retinal detachment, diaphragmatic hernias.

Question 18

Familial hypercholesteremia:

• Prevalence
• Pathogenesis
• Heterozygote Vs. Homozygote
  *

Answer 18

• 1:500
• Is the result of mutation in the gene that encodes LDL receptor. 75% of the LDL receptors are located on hepatocytes.
  
  • LDL receptor is responsible for the transport of LDL and IDL into the hepatocyte.
  • Mutation in the receptor leads to an increased serum levels of LDL and to increased conversion of IDL to LDL (→ further increase in the LDL level).
• Monocytes and macrophages have receptor for chemically modified LDL. In the case of elevated serum LDL, more binds to this scavenger receptor → appearance of skin xanthomas and premature atherosclerosis
• In the heterozygote: 2-3 times increase in the LDL level. They remain asymptomatic until adulthood, when they develop xanthomas and premature atherosclerosis which leads to coronary disease
• In the homozygote: 5 times increase in the LDL level. Develop xanthomas in childhood and die from MI at the age of 15.

Question 19

What are the groups of mutations in familial hypercholesteremia:

Answer 19

5 groups of mutations:

• Class I: no receptor synthesis
• Class II: transport from ER to Golgy is impaired
• Class III: receptor does not bind LDL
• Class IV: receptor fails to internalize
• Class V: receptor-LDL complex cannot dissociate, LDL traps in the endosome

Question 20

Characteristics of autosomal recessive disorders:

Answer 20

• both alleles on a certain locus have to be impaired
• usually parents are unaffected, but siblings may show the disease
• Siblings have 25% chance to be affected (1 out of 4)
• The expression of the defect is more uniform than autosomal dominant.
• Complete penetrance is common
• Onset is early in life
• New mutations are rare
• Disease may not show up for several generations (2 heterozygotes have to marry)
• Enzyme proteins may be affected.

Question 21

List some common autosomal recessive disorders:

Answer 21

Metabolic

• Cystic fibrosis
• phenylketonuria
• Galactosemia
• Glycogen storage disease
• Hemochromatosis
• Lysosomal storage disease

Endocrine

• Sickle cell anemia
• Thalassemia
• Hematopoietic
• Congenital adrenal hyperplasia

Skeletal

• Some variant of EDS
• Alkaptonuria

Nervous atrophies

• Spinal muscular atrophy
• Friedreich ataxia
• Neurogenic muscular

Question 22

Describe Cystic fibrosis (briefly, later it will be a separated topic)

Answer 22

CFTR gene is mutated.

Impaired Cl^- uptake of excretion → dehydration of mucus → bronchitis, bronchiectasia, pancreatitis, meconium ileum.

Question 23

Phenylketonuria

• Prevalence
• pthoigenesis
• symptoms

Answer 23

• 1:12000
• Lack of phenylalanine hydroxylase → phenylalanine cannot be converted to tyrosine → hyperphenylalaninemia → Phenylketonuria
• Affected infants are normal at birth but at the age of 6 month develop severe mental retardation, seizures, hypopigmentation of hair and skin (since tyrosine is the precursor of melanin), eczema
• Intermedietes are excreted in the urine and in the sweat: strong odor of the infant
• Restricted diet for affected individuals.

Question 24

Galactosemia (Every sing)

Answer 24

• Normally, lactose is splitted into glucose and galactose, and the later is converted to glucose in several steps.
• In this disease, galactose-1-phospate uridyltransferase is mutated → accumulation of galactose 1 phosphate and its metabolites.
• Hepatomegaly with cirrhosis, cataracts, CNS changes.
• Jaundice, vomiting, impairment of amino acid transport.

Question 25

Lysosomal storage disease

Answer 25

• With an inherited lack of lysosomal enzyme, catabolism of its substrate remains incomplete, leading to accumulation of the partially degraded insoluble metabolite within the lysosome.
• Commonly affects infants and young children
• Storage of insoluble intermediates in the mononuclear phagocytic system gives rise to splenohepatomegaly
• Neuronal damage → mental retardation
• Macrophage activation and release of cytokine lead to cellular dysfunction as a secondary event

Question 26

Tah-Sachs

Answer 26

• Gm2 gangliosidosis: deficiency in hexosaminidase α subunit
• the lack of this enzyme’s subunit lead to accumulation of Gm2 ganglioside within neurons and glial cells
• motor weakness, mental retardation, blindness, death within 2-3 years
• most common among Ashkenazi jews

Question 27

Gaucher disease

Answer 27

• mutation in the gene encoding glucosylceramidase
• gene product is responsible for cleaving glucose residues from ceramide → accumulation of glucosylceramide in the phagocytic cells → transform into Gaucher cells (large, cytoplasm is described as “wrinkled tissue paper”). Macrophages become activated and release large amounts of cytokines. There are 3 variants:
  
  • Type 1: 99% of the cases. no CNS involvement, hepatosplenomegaly and bone involvement.
  • (*?*)Compatible with life. Type 2 and 3 have CNS involvement.
• Therapy: enzyme replacement or reducing the substrate glucosylceramide by inhibiting its synthesise.

Question 28

Nieman-Pick

Answer 28

• sphyngomyelinase deficiency - sphyngomyelin storage
• Hepato-splenomegaly, mental retardation, seizures, ataxia, dysarthria

Question 29

Mucopolysaccharidoses

Answer 29

Lack of enzymes that are responsible for the degradation of mucopolysaccharides → accumulate within the lysosomes, mainly is the spleen, heart, liver and blood vessels. Coarse facial features, mental retardation, clouding of the cornea, joint stiffness.

Question 30

Glycogen storage disease

Answer 30

Due to a deficiency in one of the enzymes involved in glycogen synthesis or degradation → accumulation or abnormal form of the glycogen, in the cytoplasm, nuclei or in one case- lysosome (Pompe disease).

Types:

• Hepatic type: Von Gierke disease (type I) → lack of glucose 6 phosphatase. Hepatomegaly + hypoglycemia
• Myopathic type: McArdle’s disease (type V) → deficiency of muscle phosphorylase. Glycolysis is blocked → no energy → glycogen storage. Muscle cramps after exercise, myoglubinuria
• Pompe disease: type II → deficiency of lysosomal glucosidase → deposition in every organ but mainly in the heart

Question 31

Characteristicd of X-linked disorders

Answer 31

• Heterozygous carrier transmits to her son
• Heterozygous female rarely expresses the phenotype
• Affected male does not transmits to his son, but all his daughters are carriers.

Question 32

List some X-linked disorders

Answer 32

Musculoskeletal

• Duchenne muscular dystrophy

Blood

• Hemophilias A and B
• Chronic granulomatous disease
• Glucose 6 phosphatase dehydrogenase deficiency

Immune

• Agammaglobulinemia
• SCID
• Wiskott Aldrich syndrome

Metabolic

• Diabetes insifidus
• Lesch-nyhan syndrome

Nervous

• Fragile X syndrome

Question 33

Duchenne muscular dystrophy

• Pathogenesis
• Morphology
• Clinical features
• Cause of death

Answer 33

Pathogenesis

• Deletion in a portion of dystrophin gene, located on the short arm of the X chromosome. It is responsible for maintaining the structural and functional integrity of muscle cells by connecting a part of the sarcomere to the cell membrane.
• Analytic methods reveal that no dystorphin protein is found in individuals with DMD.

Morphology

• variation in the size of muscle fiber, due to hypertrophy and atrophy
• signs of degenerative changed (fiber splitting, necrosis)
• signs of regeneration (sarcoplasmic basophilia, nuclear enlargement, nucleolar prominence)
• adipose tissue inflitration

Clinical features

• Normal at the time of birth
• Walking is often delayed. Muscle weakness starts at the pelvic girdle and extends to the shoulder girdle, pseudohypertrophy of the calf (hypertrophy + weakness of the gastrocnemius).
• Heart failure and arrhythmias might occur, so as impairment in the CNS.
• Cause of death: respiratory insufficiency, pulmonary infection, cardiac decompensation.

Question 34

Hemophilia A

Hemophilia B

Answer 34

Hemophilia A

• Reduction in factor VIII activity
• Disease varies in degree due to the existence of different possible mutations (deletion, nonsense etc)
• X linked recessive in 75% of the cases
• Spontaneous mutation in 25% of the cases
• bleeding after minute injury
• massive hemorrhage after trauma or operative procedures
• spontaneous hemorrhages (joints → bleeding can lead to deformity)
• Therapy: infusion of facor VIII

Hemophilia B

• Defficiency of factor IX. Clinical picture is identical to type A, but it is less common.

Question 35

What are cytogenic disorders and how are they revealed?

Answer 35

• Alteration in the number or structure of chromosomes. Can affect autosomes or sex chromosomes.
• Revealed by karyotyping: photographic representation of stained metaphase chromosomes arranged in decreasing length.

Question 36

Numeric abnormalities (Names)

Answer 36

Haploid = n. normal chromosome count = 2n = 46.

Euploid: number which is an exact multiplication of n

Aneuploid: number which is not an exact multiplication of n. due to non-disjunction in the 1^st meiosis or failure of sister chromatids to separate in the 2^st meiosis. in the first case gametes are either n+1 or n-1. Fertilization of it with a normal gamete result in trisomy (2n+1) or monosomy (2n-1).

Polyploidy: 3n, 4n etc. → spontaneous abortion

Question 37

Trisomy 21

• Other name
• Reasons
• Clinical features

Answer 37

=Down syndrome

The most common cytogenic disorder. Total chromose count = 47.

Due to meiotic non-disjunction.

The frequency of the disease increases with increasing age of the mother. The extra chromosome usually comes from the mother.

Characteristic clinical features:

• epicanthic fold
• flat face
• severe mental retardation
• short neck
• congenital heart defect
• increased susceptibility for infections
• increased risk for developing acute leukemia
• risk for Alzheimer at middle age.

Question 38

Numerical aberration of sex chromosomes

Answer 38

Turner syndrome 45X karyotype. Hypogonadism

Klinefelter syndrome Male hypogonadism. Presence of at least two X chromosomes and one or more Y chromosomes. Most patients are 47 XXY.

Question 39

Turner syndrome

Answer 39

45X karyotype. Hypogonadism

Clinical features:

• growth retardation: short stature, webbing of the neck, low posterior hairline, cubitus valgus (increased carrying angle of the arms), broad chest, congenital malformations: horse-shoe kidney, bicuspid aortic valve, constriction of the aorta. Failure of development of secondary sex characteristics. Hypofunction of the ovaries, infertility, amenorrhea

Question 40

Klinefelter syndrome

Answer 40

• Male hypogonadism. Presence of at least two X chromosomes and one or more Y chromosomes. Most patients are 47 XXY.

Clinical features:

• increased length of the arms and legs, reduced hair, gynecomastia, small testicular size, wide hips. low testosterone and high gonadotropin levels

Question 41

Structural abnormalities

Answer 41

Structural changes which are the result of chromosomal breakage followed by loss or rearrangement of the material.

Type of rearrangement:

1. translocation
2. isochromosomes (horizontal, rather than ventrical, division of the centromere)
3. deletion (single or 2 breaks)
4. inversion (2 interstitial breaks → turning of the segment → reunite)
5. ring chromosome: loss of a segment from each end → unite to form a ring

p=short arm q=long arm

Each arm is devided into numbered regions, and each region contains numbered bands.

2q34 → chromosome 2, long arm, region 3, band 4.

Question 42

22q11 deletion syndromes

Answer 42

Group of disorders that include:

• congenital heart disease involving the outflow tract
• palate abnormalities
• facial dysmorphism
• developmental delay
• thymic hypoplasia (→ impaired T immunity)
• parathyroid hypoplasia (→ hypocalcemia)
• Increased risk for psychoses

Types:

• Digeorge syndrome (thymus and PT impairment)
• Velocardiofacial syndrome (face, heart)

Question 43

What are the single-gene disorders with atypical pattern of inheritance?

Answer 43

3 groups of diseases resulting from mutations affecting single genes and do not folow mendelian rules of inheritance:

• Triplet repeat mutations
• Diseases caused by mutations in mitochondrial genes
• Genomic imprinting

Question 44

What are the triplet repeat mutations?
Give one example

Answer 44

40 diseases in which there is an amplification of 3 nucleotides within a gene → function is distrupted. in all there are neurodegenerative changes.

Fragile X syndrome

Question 45

Fragile X syndrome

Answer 45

• Familial mental retardation. Abnormality in the X chromosome: a discontinuity of staining or a constriction in the long arm.
• Affected male have:
  
  • Moderate to severe mental retardation
  • Physical phenotype: long face, large mandible, large everted ears, macro orchidism (large testicles). Only the last one mentioned might be distinct.
• Cause: mutation in the FMR1 gene which maps for Xq27.3. the repeated sequence is CGG.
• Note: Permutation is a state which is between the normal repeat to the mutation (high number of repeats). Permutation is converted to full mutation by further amplification during oogenesis (not spermatogenesis!) and is then transmitted to all offsprings of the carrier female. carrier males and females have permutations. Thus →
  
  • carrier males= carry the mutation but are phenotypically and cytogenetically normal.
  • carrier females=50% of them are mentally retarded because they inherited full mutation.
• Normal repeats: 29
• Permutation: 52-200
• Full: 200-4000
• Mechanism: the CGG are located in the 5^th ‘ UTR of the FMR1 gene, and they are hypermethylated → methylation expands to the promoter region → silencing of the FMR1 gene. Its product, FMR protein (high level of expression in the testis and the brain) normally binds specific mRNAs and by that regulates its translation. The suppression of this product’s synthesis and the loss of its function is the underlying cause of this mutation.

Question 46

Diseases caused by mutations in mitochondrial genes.

• Mechanism
• What are the affected organs?
• Give an example

Answer 46

Mitochondria contains several genes that encode enzymes involved in the oxidative phosphorylation.

Ova contains many mitochondria within its cytoplasm, whereas spermatozoa contains few, it any. Thus, mitochondrial DNA of a zygote derives entirely from the mother.

Diseases in this genes affect organs most dependent on oxidative phosphorylation: skeletal** **muscle, heart, brain.

Example: Leber hereditary optic neuropathy

• (in which there is a progressive bilateral loss of central vision that leads to blindness)

Question 47

What is genomic imprinting

• Give an example

Answer 47

Each human have 2 copies of each gene: maternal and paternal.

Some genes have differences in the copies which are the result of genomic imprinting, which means gene silencing → one copy will be expressed, either maternal or paternal

• Prader Willi syndrome
• Angelman syndrome

Question 48

Describe

Prader Willi syndrome

And

Angelman syndrome

Answer 48

Prader Willi syndrome

• An interstitial deletion in the long arm of chromosome 15 coming from the father ⇒
• mental retardation, short stature, hypotonia, obesity, small palms, hypogonadism.

Angelman syndrome

• An interstitial deletion in the long arm of chromosome 15 coming from the mother ⇒ mental retardation, ataxic gait, seizues, inappropriate laugher.

These 2 diseases express the “parent of origin” phenomenon:

• if all the genes in chromosome 15 were expressed in an identical fashion, clinical features resulting from this deletions would be expected to be identical, regardeless of the parent of origin.