Diseases Caused by Mutations in Genes Encoding Proteins That Regulate Cell Growth

Diseases Caused by Mutations in MitC Genes: maternal inheritance; affect organs most dependent on oxidative phosphorylation (skeletal muscle, heart, brain). Leber hereditary optic neuropathy: progressive bilateral loss of central vision--> blindness

Frameshift mutations

Trinucleotide repeat mutations: 1. all share G and C 2.are dynamic (degree of amplification incr during gametogenesis)

missense: in β-globin chain of hemoglobin--> sickle cell anemia

nonsense: in most cases RNAs rapidly degraded (nonsense mediated decay)

miRNAs: inhibit translation of target mRNAs (Posttranscriptional silencing of gene expression); # of miRNA<<those that encode prs--> a given miRNA can silence many target genes

Autosomal Recessive Inheritance: largest group of mendelian disorders. if mutant gene occurs w/ low frequency in population--> strong likelihood of a consanguineous marriage. expression of defect tends to be more uniform than in auto dominant disorders;
Complete penetrance common. • Onset frequently early in life. • Although new mutations for recessive disorders do occur, rarely detected clinically bc affected person is an asymptomatic hetero
• In many cases, E pr are affected. In heteros, equal amounts of normal + defective E synth. U. the natural “margin of safety” ensures that cells with half of their complement of the E function normally

Autosomal Dominant: • some patients do not have affected parents (new mutations involving either egg or sperm) • Clinical features can be modified by reduced penetrance (Some persons inherit mutant gene but phenotypically normal) + variable expressivity (a trait consistently associated w/ a mutant gene but expressed differently among persons carrying it; ex: manifestations of neurofibromatosis 1 range from brownish spots on skin to multiple tumors and skeletal deformities) • In many conditions, age at onset is delayed (as in Huntington disease)

• In autosomal dominant disorders, a 50% reduction in normal gene product: clinical signs + symptom--> u. don't encode Es but instead prs:  involved in reg of complex metabolic pathways, often subject to feedback control (e.g., membrane Rs, transport proteins). ex familial hypercholesterolemia  Key structural prs, ex coll + cytoskeletal components of RBC membrane (e.g., spectrin--> hereditary spherocytosis)

biochemical mechanisms not fully understood. In some cases, esp when gene encodes one subunit of a multimeric pr: interferes w/ assembly ex: coll is a trimer (In this instance the mutant allele is called dominant negative, because it impairs the function of a normal allele) in some forms of osteogenesis imperfecta

X-Linked Disorders: (No Y-linked diseases known; Y= male
differentiation + attribute of hairy ears); Most: recessive + charac by following features: hetero females rarely express full phenotypic change; although one of X is inactivated randomly, which typically allows sufficient #s of cells w/ normal expressed allele to emerge.

B: organomegaly but no neurologic manifestations.

C: quite distinct + more common than types A + B combined. Mutations in NPC1 (most case) + NPC2: Both in transport of free cholesterol from lysosomes to cytoplasm;
Affected cells accumulate cholesterol + gangliosides
clinically heterogeneous: most common form: in childhood + marked by ataxia, vertical supranuclear gaze palsy, dystonia, dysarthria, and psychomotor regression.

Deletion: The isolated fragment, which lacks a centromere, almost never survives

Isochromosomes: centromere divides horizontally rather than vertically-->1 of 2 arms lost + remaining duplicated; most common: i(Xq) (monosomy for genes on Xp and trisomy for Xq)

ring chromosome: variant of a deletion., loss of segments from each end-->unite to form a ring.

Sex chromosomal disorders often produce subtle abnormalities, sometimes not detected at birth (infertility not diagnosed until adolescence)

In most cases, chromosomal disorders result from de novo changes (uncommon but important exception: the translocation form of Down syndrome)

Imbalances of sex chromosomes tolerated much better than autosomes

loss of chromosomal material: more severe defects than does gain

could alter gene product + predispose to a phenotypic difference / disease

Much more commonly: just a marker that is co-inherited w/ a disease-associated gene as a result of physical proximity (the SNP and the causative genetic factor are in linkage disequilibrium)--> SNPs could serve as reliable markers of risk for multigenic complex diseases (type II diabetes, hypertension,..)

complex rearrangements of genomic material, w/ multiple alleles

50% involve gene coding sequences; nificant overrepresentation of certain gene families in these regions (immune + nervous system)

development: Physiologic epigenetic silencing during development: imprinting

methylation of cytosine residues at gene promoters: heavily methyl: inaccessible to RNA polymerase

Histone pr: variety of reversible modifications (e.g., methylation, acetylation)--> secondary and tertiary DNA structure--> gene transcription

small interfering RNAs (siRNAs): Unlike miRNA, siRNA precursors are introduced by investigators into the cell. Their processing by Dicer and functioning via RISC are essentially similar to that described for miRNA.

many mechanisms, ex:bind to regions of chromatin, restricting access of RNA polymerase

XIST: tanscribed from the Xchromosome, essential role in physiologic X chromosome inactivation, itsself escapes X inactivation, but forms a repressive “cloak” on the X chromosome from which it is transcribed--> gene silencing.

different polymorphisms vary in significance in one disease (ex DM1 20-30 genes implicated, 6 or 7 are most important)

Some polymorphisms common to multiple diseases of the same type, others are disease-specific

quantitative trait loci [QTLs]: show a continuous variation within, as well as across, all population groups; ex: hair color, eye color, skin color, height, and intelligence.

Environmental influences significantly modify phenotypic expression of complex traits: ex DM2 and obesity

assigning this mode: familial clustering and the exclusion of mendelian (variable expressivity and reduced penetrance of single mutant genes also may account for this phenomenon) and chromosomal modes of transmission;

centric fusion type/robertsonian translocation:involving 2 acrocentric chromosomes; typically occur close to centromere--> one very large chromosome + one extremely small one (are lost, carry highly redundant genes (e.g., rRNA genes) ) + carrier has 45 chromosomes

median age death=47; molecular basis:elusive, incr gene dosage of pr coding genes on chromosome 21 /of effects of deregulated miRNA expression?, top culprits: DYRK1A(serine threonine kinase) + RCAN1(reg of calcineurin1)

95%: 47 chromosomes; most common cause: meiotic nondisjunction

4%: translocation of q arm of chromosome 21 to chromosome 22 or 14

1%: mosaics; mitotic nondisjunction during an early stage of embryogenesis, frequently (but not always) familial, typically robertosonian translocation in carrier parent

diagnostic: flat facial profile, oblique palpebral fissures, epicanthic folds, mental retardation (805 IQ: 25-50)

40%: congenital heart disease, most commonly defects of the endocardial cushion, (atrial septal defects, AV valve malformations, and VSDs); majority of deaths in infancy and early childhood; other congenital malformations: atresias of esophagus and small bowel also common

10-20x risk of developing acute leukemia (lymphoblastic + myeloid)

all >age 40 develop neuropathologic changes characteristic of Alzheimer disease

abnormal immune responses-->predispose to serious infections, particularly of lungs + to thyroid AI

charac: mental retardation, short stature, hypotonia, obesity, small hands and feet, hypogonadism

charac: mental retardation, ataxic gait, seizures, inappropriate laughter.

w/o a detectable cytogenetic abnormality: FISH analysis --> smaller deletions within same region

both copies of chromosome 15 derived from mother. (Inheritance of both chromosomes of a pair from one parent: uniparental disomy)

Angelman syndrome gene (UBE3A): encode a ligase in ubiquitin-proteasome proteolytic pathway; expressed primarily in specific regions of normal brain—hence the neurologic manifestations.

loss of function of several genes located on chromosome 15 btw q11 and q13

distrib: of 1 in 1550: affected males + 1 in 8000: affected females; is 2nd most common genetic cause of mental retardation, after Down syndrome

males: moderate to severe mental retardation. physical: (not always present / may be quite subtle): long face w/ large mandible, large everted ears, (in at least 90% of postpubertal males): large testicles (macroorchidism).

small amount of genetic information on Y chromosome: male differentiation; gene for male differentiation (SRY, sex-determining region of Y chromosome) on short arm

lyonization of X chromosomes: in females, only one X chromosome genetically active (P/M random) (21% of genes on Xp + 3% on Xq, escape X inactivation). X inactivation occurs early in fetal life (~16 days after conception); females are mosaics;

charac: in some only hypogonadism, but most: distinctive body habitus w/ incr in length btw soles + pubic bone--> elongated body. Also: eunuchoid body habitus, Reduced facial, body, pubic hair, gynecomastia, testes sometimes only 2 cm--> serum testosterone levels <normal + urinary gonadotropin levels
elevated.

cause: at least two X + >=1 Y. Most: 47,XXY (results from nondisjunction of sex chromosomes during meiosis, extra X M/P origin. ~15%: mosaic patterns (46,XY/47,XXY, 47,XXY/48,XXXY) 46,XY line in mosaics: milder

most common cause of hypogonadism in males; rarely fertile (mosaics w/ large proportion of 46,XY cells); cause: impaired spermatogenesis, sometimes total azoospermia. (hyalinization of tubules, Leydig cells prominent (hyperplasia / apparent incr related to loss of tubules); may be associated w/ mental retardation (typically mild, and in some not detectable) correlated w/ # of extra X; associated w/ higher frequency of several disorders (breast cancer (20x normal males), extragonadal germ cell tumors, and AI diseases like SLE)

charac: (diagnosis:at birth/early in childhood) primary hypogonadism Fig 6-16; CardioV abnormalities most common cause of death in childhood; mental status u. normal, but subtle defects in nonverbal, visual-spatial info processing; 50% develop clinical hypothyroidism. In adults: short stature + primary amenorrhea: strong suspicion for Turner; diagnosis established by karyotyping; mosaics / deletion variants may have almost normal appearance + present only w/ primary amenorrhea.

pathogenesis: (normal fetal: ovaries have 7 million oocytes; by menarche: 400,000; menopause: <10,000 remain) In Turner: absence of second X: accelerated loss of oocytes, complete by age 2yrs--> “menopause occurs before menarche,” + streak ovaries); short stature homeobox (SHOX) gene at Xp (remain active in both X + unique in having an active homologue on short arm of Y)-->1 copy: short stature.

DiGeorge syndrome: T cell immunodeficiency + hypocalcemia are dominant features; TBX1 loss correlate w/ this

velocardiofacial syndrome: mild immunodeficiency but pronounced dysmorphology + cardiac defects

diagnosis established only by FISH

are at particularly high risk for psychoses such as schizophrenia + bipolar

Cellular dysfunctions bc: storage of undigested material + cascade of secondary events triggered (ex MQ* + release of cytokines)

Storage of insoluble intermediates in mononuclear phagocyte system--> to hepatosplenomegaly

molecular basis: if misfolded prs not stabilized by chaperones-->apoptosis--> molecular chaperone therapy: use of small molecules that incr chaperone synth / reduce degradation of misfolded prs by proteosomes

most common acute infantile variant: appear normal at birth, but motor weakness begins at 3-6 mths, followed by neurologic impairment, onset of blindness, and progressively more severe neurologic dysfunctions. Death: within 2 or 3 yrs

most common among Ashkenazi Jews; carriers reliably detected by estimation of level of hexosaminidase in serum / by DNA analysis

brain principally affected bc it is most involved in ganglioside metabolism; storage of GM2: neurons, axon cylinders of nerves, glial cells throughout CNS (including the spinal cord), peripheral nerves, autonomic nervous system; retina u. involved as well, swollen ganglion cells in peripheral retina--> pallor--> contrasting “cherry red” spot in relatively unaffected central macula

bc of high phagocytic content--> organs most severely affected are spleen, liver, bone marrow, lymph nodes, and lungs. striking splenic enlargement, entire CNS involved (neurons enlarged + vacuolated).

manifests itself in infancy w/ massive visceromegaly + severe neurologic deterioration

accumulates in all phagocytic cells + in neurons. Electron microscopy: vacuoles are engorged secondary lysosomes often containing membranous cytoplasmic bodies resembling concentric lamellated myelin figures ("zebra” bodies)

accum in mononuclear phagocytic cells--> Gaucher cells particularly in liver, spleen, lymph nodes, and bone marrow.

cause: burden of storage material + *MQs (High levels of MQ-derived cytokines, (IL-1, IL-6, TNF) found in affected tissues)

type I (chronic nonneuronopathic form): 99% of cases
charac: clinical or radiographic (Marrow replacement + cortical erosion may produce visible skeletal lesions + reduction in formed elements of blood) bone involvement (osteopenia, focal lytic lesions, and osteonecrosis; caused by MQ cytokines) in 70-100% of cases + hepatosplenomegaly (spleen fills entire abdomen) + absence of CNS involvement; most common in Ashkenazi Jews; unlike other variants, compatible w/ long life.

type II (acute infantile neuronopathic form): more severe type III (chronic neuronopathic form): appear later + milder Although liver + spleen also involved, clinical features in types II and III dominated by neurologic disturbances (convulsions, progressive mental deterioration)

diagnosis + detection of carriers: level of glucocerebrosides in leukocytes / cultured fibroblasts

current: lifelong E replacement by infusion of recombinant glucocerebrosidase.

newer: reducing substrate (glucocerebroside) by oral administration of drugs that inhibit glucocerebroside synthase.

on the horizon: glucocerebrosidase gene therapy (infusion of autologous HCS cells transfected w/ normal gene)

Accumulation of dermatan + heparan sulfate in cells of mononuclear phagocyte system, fibroblasts, and within endothelium + smooth muscle cells of vascular wall--> swollen + clear cytoplasm (accumulation of material + for periodic acidSchiff staining within engorged, vacuolated lysosomes) Lysosomal inclusions also found in neurons--> mental retardation.

Hepatosplenomegaly, skeletal deformities, lesions of heart
valves, and subendothelial arterial deposits (particul in coronary arteries), and lesions in brain, in all of the MPSs. In many of more protracted syndromes, coronary subendothelial lesions--> myocardial ischemia--> myocardial infarction + cardiac decompensation important causes of death. Most cases: coarse facial features, clouding of the cornea, joint stiffness, and mental retardation. Urinary excretion of accumulated MPS often incr.

-in converting Gal to Glc: Gal-1phosphate uridyltransferase (GALT) homozygous mutations--> Gal-1-phosphate and other metabolites (galactitol) accumulate in many tissues, including liver, spleen, lens of the eye, kidney, and cerebral cortex. The liver, eyes, and brain bear the brunt

-early-onset hepatomegaly is due largely to fatty change, but in time widespread scarring closely resembling cirrhosis of alcohol abuse may supervene
-Opacification of lens (cataract), probably bc lens absorbs water + swells as galactitol accumulates + incr its tonicity.
-Nonspecific alterations in CNS: loss of nerve cells, gliosis, and edema.

-Almost from birth, affected infants fail to thrive. Vomiting + diarrhea appear within few days of milk ingestion. Jaundice + hepatomegaly usually evident during 1st week. aminoaciduria. Fulminant Escherichia coli septicemia w/ incr frequency.

-diagnosis:
demonstration in urine of a reducing sugar other than Glc, but tests that directly identify deficiency of transferase in leukocytes + RBCs. Antenatal diagnosis: assay of GALT in amniotic fluid cells / determination of galactitol level in amniotic fluid supernatant.

prevention / amelioration: early removal of Gal from diet for at least 1st 2 yrs: permits almost normal development nonetheless older patients frequently affected by a speech disorder + gonadal failure (esp premature ovarian failure) and, less commonly, by an ataxic condition.

inborn error of metabolism (inability: phenylalanine-->tyr; In normal children: <50% intake of phenylalanine necessary for pr synth, remainder converted to tyrosine by phenylalanine hydroxylase system) that affects 1 in 10,000 live-born white infants.

classic phenylketonuria: most common form quite common in persons of Scandinavian descent + distinctly uncommon in African American + Jewish populations.
-Homozygotes: classically have a severe lack of E phenylalanine hydroxylase (PAH)--> hyperphenylalaninemia + PKU.

infants normal at birth--> within few ws: rising plasma phenylalanine level--> impairs brain development, by 6 mths: severe;

-<4% of untreated phenylketonuric children have (IQs)> 50 or 60. 1/3: can't walk 2/3: can't talk. Seizures, other neurologic abnormalities, decr pigmentation of hair and skin, eczema often accompany mental retardation in untreated children.

-prevention: restriction of phenylalanine intake early in life;

maternal PKU: many females w/ PKU reach child-bearing age + clinically normal--> Most have hyperphenylalaninemia, bc dietary treatment is discontinued after reaching adulthood--> Btw 75-90% of children born to such women: mentally retarded + microcephalic, +15% have congenital heart disease, even though they're heterozygotes.
-results from teratogenic effects of phenylalanine/metabolites; presence + severity directly correlate w/ maternal phenylalanine level

-When phenylalanine metabolism blocked
-minor shunt pathways--> several intermediates excreted in large amounts in urine + sweat--> strong musty / mousy odor to affected infants.
-lack of tyrosine (a precursor of melanin)--> light hair+ skin

mutations: 500 mutant alleles of PAH gene been identified, only some cause severe deficiency; lack of PAH activity: classic features of PKU; those w/ 6% residual activity present with milder disease; some result in only modest elevations of blood phenylalanine levels w/o associated neurologic damage= benign hyperphenylalaninemia

molecular diagnosis not feasible + measurement of serum phenylalanine levels is necessary to differentiate benign from PKU; levels in latter typically are 5x (or more) higher than normal.
98% of cases of PKU: mutations in PAH; 2%: cofactor tetrahydrobiopterin (patients can't be treated by dietary restriction)

Clinical Course:
extremely varied; range from mild to severe, from presence at birth to onset years later, and from confinement to one organ system to involvement of many
Advances in management: median life expectancy: 36 yrs

milder cases: only accum of mucus in small ducts, w/ some dilation of exocrine glands.

advanced (u. seen in older children/adolescents) : ducts totally plugged--> atrophy of exocrine glands + progressive fibrosis--> impairs fat absorption--> so avitaminosis A--> squamous metaplasia of lining epithelium of ducts in pancreas, which are already injured by inspissated mucus secretions.

Intestines: Thick mucus plugs may be found in small intestine of infants; Sometimes--> small bowel obstruction (meconium ileus)

pulmonary: most serious complications; bronchioles often distended w/ thick mucus + marked hyperplasia/trophy of mucus-secreting cells (submucosal glands). Superimposed infections--> severe chronic bronchitis + bronchiectasis; Development of lung abscesses common; Staph aureus, H influenzae, and Pseudomonas aeruginosa: 3 most common organisms responsible for lung infections. Even more sinister: incr frequency of infection w/ another pseudomonad, Burkholderia cepacia: particularly hardy + associated w/ fulminant illness (“cepacia syndrome”).

Liver: Bile canaliculi plugged + ductular prolif + portal inflammation; Hepatic steatosis common; cirrhosis develops--> diffuse hepatic nodularity (in <10%)

Azoospermia + infertility in 95% of males surviving to adulthood; bilateral absence of vas deferens frequent (In some this may be the only feature suggesting an underlying CFTR mutation)

most common lethal genetic disease affecting whites; uncommon among Asians (1 in 31,000 live births) + African Americans (1 in 15,000 live births).

autos recessive + doesn't affect carriers; however bewildering phenotypic variation resulting from diverse mutations in CF-associated gene, tissue-specific effects of loss of this gene’s function, + influence of newly recognized disease modifiers.

a disorder of epithelial transport affecting fluid secretion in exocrine glands + epithelial lining of respiratory, GI, and reproductive tracts--> abnormally viscid mucous secretions blocking airways + pancreatic ducts--> 2 most imp: recurrent and chronic pulmonary infections + pancreatic insufficiency

exocrine sweat glands structurally normal (and remain so throughout the course of this disease) but a high level of NaCl in sweat is a consistent + characteristic biochemical abnormality in CF.

Class II: most prevalent form, R pr synth, but its transport from ER to Golgi impaired due to defects in pr folding.

Class III: Rs transported to cell surface but fail to bind LDL normally.

Class IV: Rs fail to internalize within clathrin pits after binding to LDL

Class V: Rs bind LDL + internalized but trapped in endosomes because dissociation of R + bound LDL does not occur.

Hetero: 2-3x elevation of plasma cholesterol levels; although cholesterol levels elevated from birth, remain asymptomatic until adult life, when they develop cholesterol deposits (xanthomas) along tendon sheaths + premature atherosclerosis.

homo: 5x; develop cutaneous xanthomas in childhood + often die of myocardial infarction <age of 20.

among most common mendelian disorders; freq: 1 in 500; mutation in LDLR gene--> accumulation of LDL cholesterol in plasma (reduced catabolism) + impairs transport of IDL into liver (--> greater proportion of plasma IDL converted into LDL (excessive biosynthesis))--> excessive levels of serum cholesterol--> marked incr of cholesterol traffic into monocyte-MQs + vascular walls mediated by scavenger R--> skin xanthomas + premature atherosclerosis.

diseases charac by defects in collagen synth / structure. All single-gene disorders auto dominant + recessive (~30 distinct types of coll--> clinical heterogeneity; at least 6 variants recognized)

certain clinical features common to all variants: tissues rich in coll (skin, ligaments,joints,..) frequently involved in most; skin is hyperextensible + joints are hypermobile--> contortions (bending thumb backward to touch forearm + bending knee upward to create almost a right angle); predisposition to joint dislocation; skin is extraordinarily stretchable, extremely fragile, and vulnerable to trauma. Minor injuries: gaping defects, and surgical interventions w/ great difficulty because of lack of normal tensile strength. serious internal complications: rupture of colon and large arteries (vascular EDS); ocular fragility, w/ rupture of cornea + retinal detachment (kyphoscoliotic EDS); and diaphragmatic hernias (classical EDS), ...

• Kyphoscoliotic EDS: Deficiency of lysyl hydroxylase: in types I + III coll, interferes w/ formation of cross-links; auto recessive

• Vascular EDS: Deficient synth of type III coll resulting from mutations affecting COL3A1 gene; auto dominant; weakness of tissues rich in type III coll (e.g., blood vessels, bowel wall), predisposing them to rupture.

• Classical EDS: Deficient synth of type V coll due to mutations in COL5A1 and COL5A2; auto dominant

Skeletal abnormalities: most obvious feature; slender, elongated habitus w/ abnormally long legs, arms + fingers (arachnodactyly); a high-arched palate; and hyperextensibility of joints. A variety of spinal deformities, such as severe kyphoscoliosis, may be present. chest: either pectus excavatum (i.e., deeply depressed sternum) or a pigeon-breast deformity.

ocular: most characteristic ocular change: bilateral dislocation, or subluxation, of the lens secondary to weakness of its suspensory ligaments (ectopia lentis).

Most serious: cardiovascular system. Fragmentation of elastic fibers in tunica media of the aorta--> aneurysmal dilation + aortic dissection. These changes, called cystic medionecrosis, not specific for Marfan syndrome, similar lesions occur in hypertension + aging. cardiac valves, esp mitral, may be excessively distensible + regurgitant (floppy valve syndrome)--> mitral valve prolapse + congestive cardiac failure. Death from aortic rupture may occur at any age + is in fact most common cause of death. Less commonly, cardiac failure is the terminal event.

auto dominant (mutant fibrillin pr must act as a dominant negative by preventing assembly of normal microfibrils); (GP that's a major component EC matrix; Microfibrils: scaffolding for deposition of tropoelastin)
particularly abundant in aorta, ligaments, and ciliary zonules that support the ocular lens; prominently affected in Marfan syndrome.

Mutations in FBN1 gene (15q21) found in all patients. However, molecular diagnosis not yet feasible, bc >600 distinct causative mutations in the very large FBN1 gene have been found.

prevalence: 1 per 5000. (70-85% familial, rest are sporadic, mutations in germ cells of parents)

some features ex overgrowth of bones difficult to relate to simple loss of fibrillin.
loss of microfibrils--> abnormal + excessive activation of TGFβ (normally sequestered)--> deleterious effects on vascular smooth muscle development + integrity of EC matrix (mutations in TGF-β type II R--> a related syndrome, Marfan syndrome type 2 (MFS2)); angiotensin R blockers, which inhibit activity of TGF-β, improve aortic + cardiac function in mouse models of Marfan syndrome

Linkage Analysis and Genome-Wide Association Studies:

In several diseases that have a genetic basis, direct genetic diagnosis is not possible (either bc causal gene not identified / disease is multifactorial (polygenic))-->2 types of analyses: linkage analysis + genome-wide association studies (GWASs). (In both, marker loci must be used to localize chromosomal regions of interest, based on their linkage to one or more putative disease-causing genes; marker loci used: polymorphisms: most common: SNPs (f: 1 n in 1kbp + found throughout genome (e.g., in exons + introns + reg sequences). SNPs: physical landmark within genome + a genetic marker whose transmission can be followed from parent to child.

enabled application of SNPs to high-throughput “gene hunting”:

1. HapMap project: provided linkage disequilibrium patterns in 3 major ethnoracial groups, based on genome-wide SNP mapping. entire human genome can now be divided into “haplotypes,” which contain varying numbers of contiguous SNPs on the same chromosome that are in linkage disequilibrium and hence inherited together as a cluster. --> comparable info about shared DNA can be obtained simply by looking for shared haplotypes, using single or a small number of SNPs that “tag” or identify a specific haplotype.

   
   

2. now possible to simultaneously genotype a million SNPs at one time, in a cost-effective way, using high-density SNP chip technology.

   
   

   • Linkage analysis: assessing shared marker loci in family members exhibiting disease or trait of interest, w/ assumption that SNPs in linkage disequilibrium w/ disease allele are transmitted through pedigree--> define a “disease haplotype” --> localization + cloning of disease allele. Linkage analysis is most useful in mendelian disorders related to one gene w/ profound effects and high penetrance.

   
   

   • polygenic: GWASs, large cohorts of patients w/ + w/o a disease (rather than families) examined across entire genome for variant SNPs that are overrepresented in persons w/ disease-->identifies regions of genome that contain a variant gene/genes confering disease susceptibility--> “candidate gene” approach (genes selected on basis of how tightly they are associated w/ the disease + whether their biologic function seems likely to be involved); GWASs also: identification of genetic loci that modulate common quantitative traits in humans, such as height, body mass, hair and eye color, and bone density.

Chromosomal level: Karyotype analysis of chromosomes by G banding: classic approach

FISH: DNA probes recognizing sequences specific to chromosomal regions>100 kb; probes labeled w/ fluorescent dyes + applied to metaphase spreads / interphase nuclei;

ability of FISH to circumvent need for dividing cells is invaluable when a rapid diagnosis is warranted, such analysis can be performed on prenatal samples (e.g., cells obtained by amniocentesis, chorionic villus biopsy, or umbilical cord blood), peripheral blood lymphocytes, and even archival tissue sections;

Use: detection of numeric abnormalities of chromosomes
(aneuploidy); subtle microdeletions or complex translocations not detectable by routine karyotyping; for analysis of gene amplification (e.g., NMYC amplification in neuroblastomas); and for mapping newly isolated genes of interest to their chromosomal loci.

Array-Based Genomic Hybridization:

unlike FISH doesn't need previous knowledge of what aberrations may be

Newer generations of microarrays using SNPs provide even higher resolution (with more than 1 million SNPs from the human genome on a single microarray!) and are currently being used to uncover copy number abnormalities in a variety of diseases, from cancer to autism

add fluorescently labeled nucleotides C and T to PCR mixture, which are complementary to either the wild-type (G) or mutant (A) sequence, respectively.

DNA sequenced + compared w/ a normal (wild-type) sequence.

Sheer volume of sequencing data (more than 1 giga–base pairs of DNA per day!) at relatively cheap costs (entire human genome has a little over 3 gigabases) In contrast with Sanger sequencing, NextGen sequencing technologies utilize platforms where sequencing of multiple fragments of the human genome (DNA or cDNA) can occur in parallel (“massively parallel sequencing”), significantly enhancing its speed ; Deep sequencing: somatic mutations in some of the most common tumor types, while germline sequencing: genetic basis of rare mendelian disorders.

Prenatal: all patients who are at risk of having cytogenetically abnormal progeny. Some important indications:
• maternal age (>34 yrs): greater risk of trisomies
• Confirmed carrier status for a balanced reciprocal translocation, Robertsonian translocation, or inversion
• A chromosomal abnormality affecting a previous child
• Determination of fetal sex when patient or partner is a confirmed carrier of an X-linked genetic disorder

Postnatal: usually performed on peripheral blood lymphocytes. Indications:
• Multiple congenital anomalies
• Unexplained mental retardation and/or developmental delay
• Suspected aneuploidy (e.g., features of Down syndrome)
• Suspected unbalanced autosome (e.g., Prader-Willi syndrome)
• Suspected sex chromosome abnormality (e.g., Turner syndrome)
• Suspected fragile X syndrome
• Infertility (to rule out sex chromosome abnormality)
• Multiple spontaneous abortions (to rule out the parents as carriers of balanced translocation; both partners should be evaluated)