Medical Genetics:Single Gene Disorders

Lecturer: David Saffen. Ph.D.Laboratory for Molecular Neuropsychiatric Genetics

Department of Cellular and Genetic MedicineSchool of Medicine, Fudan University

saffen@fudan.edu.cn

Outline

A. Historical Background

B. Patterns of inheritance

C. Genetic mutations

D. Prenatal diagnosis

A. Historical Background• Gregor Mendel

• Archibald Garrod • Thomas Hunt Morgan

• Linus Pauling

Gregor Mendel

1822-1884; German-speakingAugustinian friar in Brno, Silesia

(currently Czech Republic)

Established the rules of inheritance of discrete traits in plants, providing the foundation for the discovery of the gene and the establishment of

the modern science of genetics

Mendel’s Laws:Law of Segregation:Each individual possesses two factors (alleles) for a given trait, which “segregate” during the formation of gametes in such a manner that each gamete containsonly one of the factors (alleles). Progeny subsequentlyreceive one factor (allele) from their father and onefactor (allele) from their mother

Law of Independent Assortment:Factors (alleles) for distinct traits are inherited independently, i.e. the inheritance of a factor (allele) for one trait does not Influence the inheritance of a factor (allele) for a second trait.

Archibald Garrod

1857- 1936; British physicianProfessor Oxford University;Published Inborn Errors of

Metabolism” in 1908

Postulated that recessive metabolic disorders resulted from the inheritance of a defective enzyme from each parent.

Garrod’s tetrad: alkaptonuria, cystinuria, pentosuria, albinism

Urine samples from patientwith alkaptonuria. Left (freshlyvoided); right (after 24 h at RT)

Albinism(caused by a deficiency of tyrosinase, a enzyme

required for the synthesis of melanin)

Cystinuria(caused by defects in the SLC3A1 and SLC7A9 transporter genes)

Xylulose accumulates in the urineof patients with pentosuria.

(caused by L-xylulase deficiency)

Thomas Hunt Morgan

Link with Fudan

University:

1866- 1945; American GeneticistProfessor Columbia and California

Institute of Technology;Nobel Prize: Physiology or Medicine 1933

Working with Drosophila, established thatgene are carried on chromosome and form the

functional basis of heredity: essentially establishing the modern science of genetics

Tan Jiazhen(谈家桢 )

“Father of moderngenetics in China”

1909-2008

Linus Pauling First to demonstrate of the molecular basis

of a genetic disease, sickle cell anemia: essentially establishing the field of molecular genetics

1901-1994; American quantum chemist and biochemist.

Professor at the California Instituteof Technology & Stanford University

Nobel Prizes: Chemistry 1954; Peace 1962; Published:

Itano I et al, “Sickle Cell Anemia, a Molecular Disease”

Science 110, 1488-1490, 1949 Electrophoresis of CO-hemoglobin isolated from normal Individual (a) or individuals heterozygous (b) or

homozygous (c) for hemoglobin mutation.

Sickle-like appearance of red blood cell isolated from sickle cell anemia patient (left) compared to normal

red blood cell (right).

B. Patterns of inheritance within pedigrees

Autosomal dominant Penetrance and expressivity Autosomal recessive Consanguinity and inbreeding Co-dominant Incompletely dominant X-linked dominant X-linked recessive Y-linked

Probands and

pedigrees

Sperm

A-allele

Sperm

a-allele

Eggs

A-allele

AA aA

Eggs

a-allele

Aa aa

“Punnett squares”

Sperm

A-allele

Sperm

a-allele

Eggs

A-allele

AA aA

Eggs

a-allele

Aa aa

A = normal (major) allele a = mutated (minor) allele affected

A a

Genotype = combinationof alleles at a specific locus

unaffected

Autosomal dominant inheritance

Sperm

A

Sperm

a

Eggs

A

AA Aa

Eggs

A

AA Aa

A = normal (major) allele a = mutated (minor) allele

1/2 of offspringaffected

Sperm

A

Sperm

a

Eggs

A

AA aA

Eggs

a

Aa aa

3/4 of offspringaffected

Sperm

a

Sperm

a

Eggs

A

aA aA

Eggs

a

aa aa

all offspringaffected

Sperm

a

Sperm

a

Eggs

a

aa aa

Eggs

a

aa aa

all offspringaffected

Sperm

a

Sperm

a

Eggs

A

aA aA

Eggs

A

aA aA

all offspringaffected

Sperm

A

Sperma

Eggs

A

AA Aa

Eggs

A

AA Aade novo mutationinherited fromfather

1/2 of offspringaffected

1/2 of offspringaffected

A = normal (major) allele a = mutated (minor) allele

Example: Neurofibromatosis type 1 (NF1) An autosomal dominant disorder caused by

inactivation of one copy of the neurofibromin gene (NF1) located at 17q11.2.Incidence of NF1 = ~1/3500 births; 80% of mutations are paternal in origin.

Aa AA

Aa AaAA AA

Penetrance and expressivityPenetrance: the probability that a genetic variant will have a detectable phenotype (this is an all-or-nothing classification): Variable penetrance: describes variants with less than 100% penetrance

Expressivity: the severity of expression of the phenotypeVariable expressivity: describes phenotypes that vary among individualswith the same genotype

Lisch nodules(iris hamartomas)

Café-au-lait spots(black arrows)And cutaneousneurofibroma(white arrow)

Disseminatedcutaneous and subcuntaneousneurofibromas

Distended eye due topressure from tumor

on optic nerve

Expressivity of neurofibromatosis type 1 phenotype

Autosomal recessive inheritance

Sperm

A

Sperm

a

Eggs

A

AA aA

Eggs

a

Aa aa

A = normal (major) allele a = mutated (minor) allele

1/4 of offspringaffected

Sperm

A

Sperm

a

Eggs

a

Aa aa

Eggs

a

Aa aa

1/2 of offspringaffected

Sperm

a

Sperm

a

Eggs

a

aa aa

Eggs

a

aa aa

all offspringaffected

Aa Aa

aa aa

Example: Cystic fibrosis (CF) An autosomal recessive disorder caused by mutations in the CF

transmembrane regulator gene (CFTR) located at 7q31.2.Large differences in frequency of CFTR mutations in different populations:

~1/25 among Northern Europeans; ~1/500 in Asian populations

Symptoms include: abnormal chloride ion transport in exocrine tissues leadingto the accumulation of mucus in lungs, sinuses, intestines, pancreas and malereproductive tract. Accompanying bacterial infections and inflammation cause

tissue damage and organ failure. Expression of CF symptoms is highly variable.

Lung from cysticFibrosis patient

CBAVD = congenital absence of

vas deferens

Consanguinity and inbreeding

F = probability that a homozygote has

received both allelesat a locus from the same ancestor; i.e. that the alleles are

identical-by-decent (IBD)*

Probabilities that an individualharbors one or two copies

of the A1 allele

1.0 0.0

0.50.5

0.250.25

0.0625 = 1/16

*Note: alleles that produce the same phenotype, but at not known to be IBD are termed identical-by-state (IBS).

aa

AaAa

Aa Aa

A rare, autosomal recessive disorder caused by mutations in genes encoding enzymes required for repair of ultra violet light-damaged DNA. Eight subtypes have been defined,

based upon mutations in different genes. The classical form is caused by mutations in the XP complementation group A gene (XPA) located at 9q22.3, which encodes a “zing finger” protein required for nucleotide excision repair. The inability to repair damaged DNA gives rise to malignant melanoma and

basal cell and squamous cell carcinomas.

Prevalence in US and Europe: ~1/1,000,000; prevalence in Japan ~1/100,000; 20% of cases derive from offspring of

marriages between first cousins.

Example: Xeroderma pigmentosum (XP)

Co-dominant inheritance

Example: the ABO blood types are determined by three variants of the alpha 1-3-galactosyltransferase gene (ABO), located at 9q34.2. The “A” variant produces the “A” antigen on the surface of red

blood cells by adding an N-acetylgalactosamine residue to a cell surface glycoprotein called H-antigen. Similarly, the “B” variant produces “B” antigen

by adding a D-galactosamine residue to H-antigen. By contrast, the “O” variantis inactive, resulting in red blood cells with unmodified H-antigen on the cell surface.

Variants A and B are dominant with respect to the O variant, but co-dominant with respect to each other.

A B O

A AA AB AO

B AB BB BO

O AO BO OO

A

B

AB

O

Incompletely dominant inheritance[homozygotes more severely affected than heterozygotes]

Note: when modeling the contributions of genetic variants to a disease, the variants areoften assumed to have additive effects: homozygotes carrying a liability variant are

assumed to be roughly two-fold more severely affected than heterozygotes ortwo-fold more likely to be diagnosed with the disease.

Example: Acondroplasia, the most common form of dwarfism, is caused by specific mutations in the fibroblast growth factor

receptor subtype 3 gene (FGFR3), located at 4p16.3. Two mutations: 1138G>A (~98%) and 1138G>C (~1-2%) account for 99% of

cases. These are gain-of-function mutations that change the amino acid sequence of FGFR3

In such a way that causes the receptor tobecome constitutively active.

The incidence of acondroplasia is 1/15,000 to 1/40,000 in all ethnic groups. De novo mutationOf 1138G occur exclusively in the paternal germLine and the frequency increases with age (>35 years). Homozygous achondroplasia is lethal.

X-linked dominant inheritance with male lethality

Disorder is observed in ~50% of daughters of affected mothers;Males die during prenatal period. Expressivity in affected females

Varies with underlying pattern of X-inactivation

Example: Rett syndromeXq28: methyl CpG binding protein 2 gene (MECP2) gene

Prevalence among females: 1/10,000 – 1/15,000. rarely observed in 47, XXY males.Mutations in MEPC2 are thought to interfere with transcriptional silencing and

epigenetic regulation of genes in regions of methylated DNA, leading to inappropriateactivation of gene expression. Symptoms include small brains with cortical and cerebellar

Atrophy without loss of neurons (smaller cells with fewer dendritic branches); motor disabilities; stereotypic hand-wringing and circulating hand-mouth movements, epilepsy, autism

DNMT1 = DNA (cytosine-5) methyltransferase 1HDAC = histone deacetylase

X-linked recessive inheritance

Female carrier: May be unaffectedor suffer variable symptoms depending

upon pattern of X inactivation

Example: Hemophilia A and B [Xp28 (F8=cofactor) and Xp27.1-p27.2 (F9= protease)]

Incidence: ~1/5000 (F8) and ~1/100,000 (F9) newborn malesSeverity of symptoms depends on residual Factor VIII or IX activity:

severe (<1%); moderate (1%-5%); mild (5%-25%); Clotting factor replacement has extended life expectancy from 1.4 year in early 1900’s to 65 years today.

Y-linked inheritance

Example: azoospermia caused by deletions in AZF regions of the Y-chromosome

Normal testis

Sertoli cell only testis

Locus heterogeneityExample: Charcot-Marie-Tooth Neuropathy

39 CMT risk loci

Gene

Pulmonary

Function (FEV1)

Pseudomonas aeruginosa acquisition/

colonization

Intestinal Obstruction

Diabetes Liver

Disease

ADIPOR2 +

EDNRA ++

IFRD1 +

ILS +

MBL2 ++ ++

MSRA +

SERPINA1 - - +

TCF1I2 +

TBFB1 ++ - +

Ref: Cutting GR, Modifier genes in Mendelian disorders: the example of cystic fibrosis, Ann NY Acad Sci 1214, 57-69, 2010

Disease modifier genes

Example: cystic fibrosis

Founder mutations and the risk of genetic disorders in isolated populations

Example: Tay-Sachs disease (recessive inheritance)

Multi-generational

pedigree demonstrating

common remote

common ancestorsin Cajun

population

*Heterozygous for hex A -subunit exon 11 insertion; **Homozygous for hex A -subunit exon 11 insertion

*Heterozygous for G to A transition in spice donor site in intron 9 of hex A a-subunit

*

**

****

** ** *

* ** *

*

** **

** * *

**

Maintenance of deleterious mutations in the population: Heterozygote advantage

Prevalence of Malaria (left) and sickle-cell disease (right) in Africa

Recessive genetic disorders with

suspected heterozygote advantage

Disorder

Heterozygotes resistant to:

Sickle-cell anemia Malaria

Thalassemia Malaria

Primaquine/fava bean-induced hemolysis

(glucose-6-phosphate dehydrogenase deficiency)

Malaria

Cystic fibrosis Cholera

Tuberculosis

Chronic obstructive lung disease(emphysema) cirrhosis of liver

1-antitrypsin deficiency)Tuberculosis

C. Genetic mutations

• Point mutations

Coding sequence mutations

Mutations affecting RNA splicing

Promoter mutations• Indels • Repeat expansions

Intergenerational “anticipation”

Missense mutationsExample: sickle-cell disease

Note: this is an example of again-of-function mutation

Nonsense mutations and nonsense-mediated mRNA decay (NMD)

Examples: Tryptophan-to-stop codon mutation in PAX3

and nonsense mutations in PAX10

PAX3

PAX10

NMD: complete loss-of-function Mutant “gain-of-function” proteins(Waardenburg syndrome: hearing (Severe neurological deficits)loss, pigmentation abnormalities, Hirschsprung disease)

Nonsense mutations and nonsense-mediated mRNA decay (NMD)

Examples: Tryptophan-to-stop codon mutation in PAX3

and nonsense mutations in PAX10

PAX3

PAX10

NMD: complete loss-of-function Mutant “gain-of-function” proteins(Waardenburg syndrome: hearing (Severe neurological deficits)loss, pigmentation abnormalities, Hirschsprung disease)

Mutations affecting RNA splicingExon Intron

CFTR (CF transmembrane conductance regulator)

Novel, deleterious splice site: frame-shifted aa sequence containing termination codons

*

*MITF = microphthalmia- associated transcription factor (mutations cause Waardenburg syndrome type 2)

**SMN1 survival of motor neuron 1 SMN2 survival of motor neuron 2 (mutations in SMN1 cause spinal muscular atrophy)

**

**

Promoter mutations

Example: clotting factor IX (Leyden)

Small insertions and deletions (indels) Example: Frame-shift mutation in Tay-Sachs disease

Insertion of four nucleotidesinto exon 11 of

hexosaminidase A geneshifts the reading frame and

introduces a prematurestop codon

In the absence of hex A activityGM2 ganglioside accumulates in the brain, causing neurological damage and death by age 2 to 4.

The rate of de novo mutationsincrease with father’s age

Ref: Kong A et al, Rate of de novo mutation and the importance of father’s age to disease risk, Nature 488, 471-475, 2012

Repeat expansions

Huntington disease

Age 50

Progressive and fatalneurodegenerative

disorder that producesuncontrolled movements

cognitive deficits and emotional disturbances.

All areas of the brain affected,with particularly extensive

damage to striatum (caudatenucleus and putamen)

Dominant inheritance, with symptoms typically appearing

In middle age.

Currently ~ 15,000 HD patientsIn US, with an additional 75,000

Pre-onset heterozygotes.

Proposed mechanism of repeat expansion

Inter-generational “anticipation”:progressive increase in repeat length and

decrease in age of onset in an HD pedigree

Ref: Ranen et al, American Journal Human Genetics 57, 593-6022, 1995

Catalog of Mendelian disorders

Mendelian Inheritance in Man http://www.ncbi.nlm.nih.gov/omin/

To date, causative genetic variants for approximately 3,000 Mendelian disorders have been identified

D. Prenatal diagnosis of genetic disorders: Amniocentesis and chorionic villus sampling (CVS)

Note: fetal blood can be directly obtained from the umbilical cord (cordocentesis).

Non-invasive methods for prenatal diagnosis

Note: AFP = alpha-fetoprotein

Non-invasive genetic tests

Science Translational Medicine. 2012 Jun 6;4(137):137ra76.Noninvasive whole-genome sequencing of a human fetus.

Kitzman JO, Snyder MW, Ventura M, Lewis AP, Qiu R, Simmons LE, Gammill HS, Rubens CE, Santillan DA, Murray JC, Tabor HK, Bamshad MJ, Eichler EE, Shendure J.

Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.

PLoS One. 2012;7(5):e38154. doi: 10.1371/journal.pone.0038154. Epub 2012 May 29.Noninvasive prenatal diagnosis of fetal trisomy 21 by allelic ratio analysis

using targeted massively parallel sequencing of maternal plasma DNA.Liao GJ, Chan KC, Jiang P, Sun H, Leung TY, Chiu RW, Lo YM.

Centre for Research into Circulating Fetal Nucleic Acids, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Shatin,

New Territories, Hong Kong, China.

RI Nussbaum, RR McInnes and HF Willard, “Thompson & Thompson Genetics in Medicine, Edition 7,” 2007, Saunders Elsevier, Philadelphia, PA;ISBN: 978-1-4160-3080-5 (Chapters 7 and 15)

T Strachan and A Read, “Human Molecular Genetics, 4th Edition,” 2011, Garland Science, New York, New York ISBN: 978-0-815-34149-9 (Chapter 13)

References and further reading