Human Genetics and Genetic Disorders BMEG3102 Huating Wang, Ph.D. Dept. of ORT huating.wang@cuhk.edu.hk

What is genetics? Genetics is the study of genes, and studies what genes are and how they work. Study of human genetics can be useful as it can answer questions about human nature, understand the diseases and development of effective disease treatment, understand genetics of human life.

Genetic disorder A genetic disorder is an illness caused by abnormalities in the genome, especially a condition that is present from birth (congenital). Most genetic disorders are quite rare and affect one person in every several thousands or millions. Genetic disorders are heritable, and are passed down from the parents' genes. Other defects may be caused by new mutations or changes to the DNA. In such cases, the defect will only be heritable if it occurs in the germ line. The same disease, such as some forms of cancer, may be caused by an inherited genetic condition in some people, by new mutations in other people, and by nongenetic causes in still other people. Some types of recessive gene disorders confer an advantage in certain environments when only one copy of the gene is present.[1]

Mutations Defined as a permanent change in the DNA Origin germ cells – transmitted to progeny somatic cells – cancer and some congenital malformations Types of mutation Chromosome mutation – structural changes within the chromosome – translocations, deletions, etc Genome mutation – loss or gain of whole chromosomes: monosomy and trisomy gene mutation – alterations at the level of the gene

Genetic Code

Point mutation result from substitution of a single base in the DNA Coding portion of gene Missense– result in substitution of one amino acid for another in the coded protein conservative – function of protein is not affected nonconservative – function of protein altered Nonsense stop codon – results in truncated protein Noncoding portion of gene promoter and enhancer regions posttranslational processing – defective splicing

Deletions and Insertions Deletion of multiple of 3 bases three bases code for one amino acid abnormal protein missing one or more amino acids Frameshift mutation will result in a different sequence of base triplets meaning of genetic code is altered distal to the mutation usually leads to stop codon and truncated protein

Hemoglobin S: Point Mutation Resulting From A Single Base Pair Change In The DNA (Sickle Cell Anemia)

Tay-Sachs Disease: Four-base Insertion In The Hexosaminidase A Gene

β0 Thalassemia: Point Mutation Leading To Premature Chain Termination

Three-base Deletion In The Common Cystic Fibrosis (CF) Allele

I. Single Gene Disorder The result of a single mutated gene Monogenic disease; Mendelian disorder The result of a single mutated gene Over 4000 human diseases are caused by single gene defects. Single gene disorders can be passed on to subsequent generations in several ways. Most follow pattern of Mendelian inheritance Main types Autosomal dominant Autosomal recessive X-linked dominant X-linked recessive Y-linked

Pedigree Pedigree: a family history that shows how a trait is inherited over several generations. Pedigrees are usually used when parents want to know if they are carriers of a particular disorder Carriers are heterozygous for a disorder and don’t show symptoms, BUT have the allele to pass on to their offspring

Making a Pedigree Filled in symbols indicate individual is affected with a disorder Female Male Married Couple Siblings

Example of a Pedigree Grandparents Grandparents Parents Aunts, Uncles What can you tell from a pedigree? Whether a family has an autosomal or sex-linked disease or disorder Autosomal disorder: appears in both sexes equally Sex-linked disorder: allele is located only on the X or Y chromosome. Most sex-linked genes are on the X chromosome and are recessive So who would have an X-linked disorder more often, boys or girls? Whether a disorder is dominant or recessive Brother You

Autosomal Dominant Only one mutated copy of the gene will be necessary for a person to be affected by an autosomal dominant disorder. Each affected person usually has one affected parent. The chance a child will inherit the mutated gene is 50%.  Examples of autosomal dominant traits and disorders are Huntingtons disease and achondroplasia.

Huntington’s Disease Huntington's disease (HD) is an inherited, degenerative brain disorder which results in an eventual loss of both mental and physical control. The disease is also known as Huntington's chorea. Chorea means "dance-like movements" and refers to the uncontrolled motions often associated with the disease.

cause progressive cell death. Huntington gene 1 ttg ctg tgt gag gca gaa cct gcg ggg gca ggg gcg ggc tgg ttc cct ggc cag cca ttg 61 gca gag tcc gca ggc tag ggc tgt caa tca tgc tgg ccg gcg tgg ccc cgc ctc cgc cgg 121 cgc ggc ccc gcc tcc gcc ggc gca cgt ctg gga cgc aag gcg ccg tgg ggg ctg ccg gga 181 cgg gtc caa gat gga cgg ccg ctc agg ttc tgc ttt tac ctg cgg ccc aga gcc cca ttc 241 att gcc ccg gtg ctg agc ggc gcc gcg agt cgg ccc gag gcc tcc ggg gac tgc cgt gcc 301 ggg cgg gag acc gcc atg gcg acc ctg gaa aag ctg atg aag gcc ttc gag tcc ctc aag 361 tcc ttc cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag 421 cag cag cag caa cag ccg cca ccg ccg ccg ccg ccg ccg ccg cct cct cag ctt cct cag Encodes a run of 11-34 glutamine amino acid residues in the HD protein. A run of > 34 glutamine residues causes the protein to aggregate in the brain cells and cause progressive cell death.

Autosomal Recessive Two copies of the gene must be mutated for a person to be affected by an autosomal recessive disorder. An affected person usually has unaffected parents who each carry a single copy of the mutated gene (and are referred to as carriers). Two unaffected people who each carry one copy of the mutated gene have a 25% chance with each pregnancy of having a child affected by the disorder. Examples of this type of disorder are cystic fibrosis, sickle cell disease, Tay Sachs disease, Spinal muscular atrophy and Roberts syndrome.

Sickle Cell Anemia Disorder where abnormal hemoglobin (a protein inside red blood cells) is produced and warps red blood cells Sickle cells deliver less oxygen to body’s tissues and can get stuck in small blood vessels tends to be seen in people of African or Mediterranean descent Hemoglobin carries oxygen to cells, life expectancy shorter, patients used to die of organ failure between 20 and 40, but now people tend to live into their 50s in the US anyway

Cystic Fibrosis Life threatening, causes thick mucus to build up in various areas of the body (lungs, digestive tract, etc). Tends to run in Caucasians, of Northern/Central European descent (1 in 29 Americans carry the allele) Average life span in US for people with CF is 37, death usually caused by lung complications To help, medications are often given for mucus buildup and have to keep a strict diet to help with digestion problems

X-linked recessive X-linked recessive conditions are also caused by mutations in genes on the X chromosome. Males are more frequently affected than females, and the chance of passing on the disorder differs between men and women. The sons of a man with an X-linked recessive disorder will not be affected, and his daughters will carry one copy of the mutated gene. A woman who is a carrier of an X-linked recessive disorder (XRXr) has a 50% chance of having sons who are affected and a 50% chance of having daughters who carry one copy of the mutated gene and are therefore carriers.  hemophilia A, Duchenne muscular dystrophy, and Lesch-Nyhan syndrome, male pattern baldness and red-green color blindness.

Hemophilia Bleeding disorder, where it takes a long time for blood to clot (body lacks protein FVIII involved in clotting) Sex-linked (carried on the X chromosome) Treatment involves injection with missing clotting protein. the royal disease. People born with hemophilia have little or no clotting factor. Clotting factor is a protein needed for normal blood clotting. There are several types of clotting factors. These proteins work with platelets to help the blood clot.

X-linked Inheritance pedigree chart Haemophilia has featured prominently in European royalty and thus is sometimes known as 'the royal disease'. Queen Victoria passed the mutation for Haemophilia B[35][36] to her son Leopold and, through some of her daughters, to various royals across the continent, including the royal families of Spain, Germany, and Russia. In Russia, Tsarevich Alexei Nikolaevich, son of Nicholas II, was a descendant of Queen Victoria through his mother Empress Alexandra and suffered from haemophilia. It was claimed that Rasputin was successful at treating Tsarevich's haemophilia. At the time, a common treatment administered by professional doctors was to useaspirin, which worsened rather than lessened the problem. It is believed that, by simply advising against the medical treatment, Rasputin could bring visible and significant improvement to the condition of Tsarevich. In Spain, Queen Victoria's youngest daughter, Princess Beatrice, had a daughter Victoria Eugenie of Battenberg, who later became Queen of Spain. Two of her sons were haemophiliacs and both died from minor car accidents. Her eldest son, Prince Alfonso of Spain, Prince of Asturias, died at the age of 31 from internal bleeding after his car hit a telephone booth. Her youngest son, Infante Gonzalo, died at age 19 from abdominal bleeding following a minor car accident where he and his sister hit a wall while avoiding a cyclist. Neither appeared injured or sought immediate medical care and Gonzalo died two days later from internal bleeding.

Duchenne Muscular Dystrophy Duchenne muscular dystrophy is a disease in which the muscles of the body get weaker and weaker and slowly stop working because of a lack of dystrophin protein.

Color Blindness Deficiency to perceive colors Problem with color-sensing pigments in certain nerve cells of the eye About 1 in 10 men have some form of color blindness. Can get color blind from nerve damage, or drugs (one drug used to treat arthritis causes color blindness), but most of the time is a genetic disorder. Color blindness is a life long condition, doesn’t impact day to day life that much. Color blind people may not be able to do certain jobs that require seeing color, like a fashion designer or a cook who needs to be able to tell when the meat is done, or an electrician who can’t tell the difference between wire colors that she or he needs to cut

X-linked dominant X-linked dominant disorders are caused by mutations in genes on the X chromosome. Only a few disorders have this inheritance pattern, with a prime example being X-linked hypophosphatemic rickets. Males and females are both affected in these disorders, with males typically being more severely affected than females. Some X-linked dominant conditions, such as Rett syndrome, incontinent pigment type 2 and Aicardi syndrome, are usually fatal in males either in utero or shortly after birth, and are therefore predominantly seen in females.

II. Chromosomal disease Human disorders due to chromosome alterations. Most chromosome abnormalities occur as an accident in the egg or sperm, and therefore the anomaly is present in every cell of the body. Some anomalies, however, can happen after conception. If the parents do not possess the abnormality it was not initially inherited; however it may be transmitted to subsequent generations.

Types Of Chromosomal Rearrangements Translocation: a segment from one chromosome is transferred to another Balanced: no genetic material is lost and patient is often normal, but may produce abnormal gametes. Robertsonian: translation between acrocentric chromosomes, with formation of a very large chromosome and a very small one, whose genetic material is often lost. If lost material is not critical, patient may be normal, but progeny are at risk. Isochromosome: one arm is lost and the remaining arm is duplicated. Chromosome has two short arms or two long arms. This abnormality usually involves the X-chromosome where such losses are tolerated better than in autosomes. Ring chromosomes do not behave normally in meiosis or mitosis and have serious consequences.

Aneuploidy Abnormal number of chromosomes Autosomal: Trisomy 21 (Down syndrome) Trisomy 18 (Edward syndrome) Trisomy 13 (Patau syndrome) Sex chromosome: 47XXY (Klinefelter syndrome) 45X (Turner syndrome)

Down’s Syndrome Caused by non-disjunction of the 21st chromosome. Most common chromosomal disorder Affects 1 in 750 newborns overall, but is related to maternal age 1 in 1550 live births of mothers > 20 years 1 in 25 live births of mothers > 45 years

Down’s Syndrome or Trisomy 21

Symptoms of Down Syndrome Upward slant to eyes. Small ears that fold over at the top. Small, flattened nose. Small mouth, making tongue appear large. Short neck. Small hands with short fingers.

Symptoms of Down Syndrome Low muscle tone. Single deep crease across center of palm. Looseness of joints. Small skin folds at the inner corners of the eyes. Excessive space between first and second toe. In addition, down syndrome always involves some degree of mental retardation, from mild to severe. In most cases, the mental retardation is mild to moderate.

Clinical Features of Down Syndrome

Turner’s Syndrome

Turner syndrome is associated with underdeveloped ovaries, short stature, webbed, and is only in women. Bull neck, and broad chest. Individuals are sterile, and lack expected secondary sexual characteristics. Mental retardation typically not evident.

Klinefelter syndrome 47, XXY

III. Multifactorial and polygenic (complex) disorders Associated with the effects of multiple genes in combination with lifestyles and environmental factors. heart disease, most cancers, and behavioral disorders such as alcoholism, obesity, mental illness, and Alzheimer’s disease are examples 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. On a pedigree, polygenic diseases do tend to "run in families", but the inheritance does not fit simple patterns as with Mendelian diseases. But this does not mean that the genes cannot eventually be located and studied. There is also a strong environmental component to many of them (e.g., blood pressure).

Multifactorial etiology of disease Multifactorial etiology of disease. An individual is born with a genetic liability but remains in a presymptomatic state for some time, until additional events occur, including exposure to environmental factors, that result in crossing a threshold that is identified as disease. In instances of high-penetrance monogenic disorders, the genetic liability may be overwhelming. In other instances, genetic factors may contribute only slightly to disease risk. Goldman: Cecil Textbook of Medicine, 22nd ed.

Genetic testing: One of the fastest moving fields in medical science. A technique to determine the genotype and phenotype of an organism. It is often used to detect faulty or abnormal genes in an organism.

Genetic Testing: Types of test: Prenatal diagnosis: determine genotype of fetus Newborn testing: determine genotype of newborns Carrier testing: test family members, determine chances of having an affected child

Other types of screening Preimplantation screening: Screening embryos fertilized by IVF before they are implanted into the uterus. Presymptomatic screening: Screening to predict adult-onset diseases such as Huntington’s; Screening to estimate the risk of developing cancer or Alzheimer’s disease as an adult. Forensic/Identity testing: Screening to determine the father of an individual (paternity test).

Prenatal Screening This can detect a disorder before a baby is born. An ultrasound test is used to determine if the fetus is at a high or low risk from a genetic disorder. Disorders are diagnosed by examining a small amount of fetal cells. This carries a small risk to the fetus. If diagnosed early in the pregnancy, there is still the possibility of abortion. Prenatal screening is sometimes seen as controversial.

Prenatal test: Non-invasive Ultrasound blood Invasive Amnioscentisis Chorionic villus sampling

Ultrasound Noninvasive, uses reflected sound waves converted to an image Screening tool in all trimesters See physical features of fetus, not chromosomes May ID some chromosomal abnormalities by physical features

Ultrasound of Fetus with Neck Fold (nuchal scan) Fetuses with Down syndrome tend to have an increased amount of fluid around the neck. Nuchal scanning alone detects 62% of all Down's Syndrome with a false positive rate of 5.0%.

Amniocentesis One of the most common prenatal tests A sample of amniotic fluid is removed and tested for alpha-fetoprotein level

Amniocentesis Diagnose > 100 disorders. Risk of infection and spontaneous abortion Normally only used when: Advanced maternal age History of chromosomal disorder Parent with chromosomal abnormality Mother carrier of X-linked disorder

Amniocentesis

Chorionic Villus Sampling Collection of fragments of placental tissue (chorionic villi)- Cytotrophoblastic (rapidly dividing) cells are used for direct karyotyping- result available within 24-48 h. Chorionic villi are best source of DNA CVS can be performed at 10 weeks gestation. Risks: Pregnancy loss 2-6%

Procedure: Trans-abdominal approach preferred –under USG guidance in supine position Trans-cervical approach is easy. In lithotomy position, sterilize area & Aspiration catheter and biopsy forceps. Introduce through Cx. under USG into placental tissue avoiding membrane rupture Before 10 weeks- associated with limb deformities, micrognathia, microglassia

What to measure in sample? Chromosomes Protein/enzyme levels DNA-sequence

Cytogenetic/Molecular Analytic Techniques Karyotyping FISH PCR Array Comparative Genomic Hybridisation (aCGH) Sequencing

banded karyotypeing A karyotype is the number and appearance of chromosomes in the nucleus of a eukaryotic cell. Karyotypes describe the number of chromosomes, and what they look like under a light microscope. Abnormal number of chromosomes Large duplications and deletions Balanced rearrangements (translocations, inversions)

G-banding is obtained with Giemsa stain following digestion of chromosomes with trypsin. It yields a series of lightly and darkly stained bands — the dark regions tend to be heterochromatic, late-replicating and AT rich. The light regions tend to be euchromatic, early-replicating and GC rich. Figure 5-20 Normal male karyotype with G banding. (Courtesy of Dr. Nancy Schneider, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX.) Note that karyotyping requires dividing cells in order to obtain metaphase spreads of chromosomes. In interphase cells, the genetic material is dispersed and chromosomes are not visible. In normal  diploid organisms, autosomal chromosomes are present in two copies. Downloaded from: Robbins & Cotran Pathologic Basis of Disease (on 18 July 2005 09:03 PM) © 2005 Elsevier

FLOURESCENT IN SITU HYBRIDIZATION (FISH) FISH allows detection & localization of specific DNA sequence in interphase or metaphase. Advantage – rapid aneuploidy screening, results available in 24-48 h. Disadvantage – fail to detect big structural rearrangements Identify 80% clinically relevant abnormalities, helpful for early decision about further management of affected pregnancies.

FISH fluorescent in situ hybridization: (FISH) A technique used to identify the presence of specific chromosomes or chromosomal regions through hybridization (attachment) of fluorescently-labeled DNA probes to denatured chromosomal DNA. Step 1. Preparation of probe. A probe is a fluorescently-labeled segment of DNA complementary to a chromosomal region of interest. Step 2. Hybridization. Denatured chromosomes fixed on a microscope slide are exposed to the fluorescently-labeled probe. Hybridization (attachment) occurs between the probe and complementary (i.e., matching) chromosomal DNA.

FISH Step 3. Visualization. Following hybridization, the slide is examined under a microscope using fluorescent lighting. Fluorescent signals indicate the presence of complementary chromosomal DNA; absence of fluorescent signals indicate absence of complementary chromosomal DNA. Green signal = Normal control Pink signal = Chromosome region of interest Normal control: Two green signals Two pink signals Patient with deletion: Two green signals One pink signal

FISH analysis

Polymerase chain reaction (PCR) Amplify specific DNA and RNA fragments Once nucleotide sequence of a region of DNA strand is known, complimentary oligonucleotides & polymerase are added to single strand DNA Repeat process 30 times to get adequate DNA PCR identify specific DNA sequence for gene mutation.

Visualization of the DNA Polymerase chain reaction. The polymerase chain reaction (PCR) generates multiple copies of a DNA segment. After denaturing the double-stranded (ds) DNA, complementary synthetic oligonucleotide primers of about 20 bp are annealed on each side of the fragment of interest. A heat-stable polymerase then extends the oligonucleotides and synthesizes the complementary strand. This cycle is repeated 25 to 30 times. The number of DNA-amplified DNA segments is thus doubled after every PCR cycle. Gel electrophoresis a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge.

cause progressive cell death. Huntington gene 1 ttg ctg tgt gag gca gaa cct gcg ggg gca ggg gcg ggc tgg ttc cct ggc cag cca ttg 61 gca gag tcc gca ggc tag ggc tgt caa tca tgc tgg ccg gcg tgg ccc cgc ctc cgc cgg 121 cgc ggc ccc gcc tcc gcc ggc gca cgt ctg gga cgc aag gcg ccg tgg ggg ctg ccg gga 181 cgg gtc caa gat gga cgg ccg ctc agg ttc tgc ttt tac ctg cgg ccc aga gcc cca ttc 241 att gcc ccg gtg ctg agc ggc gcc gcg agt cgg ccc gag gcc tcc ggg gac tgc cgt gcc 301 ggg cgg gag acc gcc atg gcg acc ctg gaa aag ctg atg aag gcc ttc gag tcc ctc aag 361 tcc ttc cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag cag 421 cag cag cag caa cag ccg cca ccg ccg ccg ccg ccg ccg ccg cct cct cag ctt cct cag Encodes a run of 11-34 glutamine amino acid residues in the HD protein. A run of > 34 glutamine residues causes the protein to aggregate in the brain cells and cause progressive cell death.

The data below shows the results of electrophoresis of PCR fragments amplified using probes for the site which has been shown to be altered in Huntington's disease. The male parent, as shown by the black box, got Huntington's disease when he was 40 years old. His children include 6 (3,5,7,8,10,11) with Huntington's disease, and the age at which the symptoms first began is shown by the number above the band from the PCR fragment. - DNA migrates from – to + the larger the fragment is, the slower it runs. + Quiz: No relationship between age of onset of disease and the migration rate of PCR fragments. A shorter PCR fragment predicts early onset of Huntington's disease. Increased length of the amplified PCR fragment predicts early onset of Huntington's disease. Huntington's disease must be contagious since many of the children have the disease.

Population or subgroup BRCA1 and BRCA2 (breast cancer , early onset) are normally expressed in the cells of breast and other tissue, where they help repair damaged DNA or destroy cells if DNA cannot be repaired. They are involved in the repair of chromosomal damage with an important role in the error-free repair of DNA double-strand breaks. Population or subgroup BRCA1 mutation(s)[57] Reference(s) African-Americans 943ins10, M1775R [58] Afrikaners E881X [59] Ashkenazi Jewish 185delAG, 188del11, 5382insC [54][55] Austrians 2795delA, C61G, 5382insC, Q1806stop [60] Belgians 2804delAA, IVS5+3A>G [61][62] Dutch Exon 2 deletion, exon 13 deletion, 2804delAA [61][63][64] Finns 3745delT, IVS11-2A>G [65][66] French 3600del11, G1710X [67] French Canadians C4446T [68] Germans 5382insC, 4184del4 [69][70] Greeks 5382insC [71] Hungarians 300T>G, 5382insC, 185delAG [72] Italians 5083del19 [73] Japanese L63X, Q934X [74] Native North Americans 1510insG, 1506A>G [75] Northern Irish 2800delAA [76] Norwegians 816delGT, 1135insA, 1675delA, 3347delAG [77][78] Pakistanis 2080insA, 3889delAG, 4184del4, 4284delAG, IVS14-1A>G [79] Polish 300T>G, 5382insC, C61G, 4153delA [80][81] Russians 5382insC, 4153delA [82] Scottish [76][83] Spanish R71G [84][85] Swedish Q563X, 3171ins5, 1201del11, 2594delC [58][86] Test for 5382insC mutation presence (lanes 1 and 5) or absence (lanes 2 - 4). PCR amplified fragments of exon 20 of the BRCA1 gene were subjected to electrophoresis and DNA bands were visualized by silver staining.

Array Comparative Genomic Hybridisation Chromosomal microarray analysis, microarray- based comparative genomic hybridization, array CGH, a-CGH, aCGH) is a technique to detect genomic copy number variation at a high resolution.

Next generation sequencing

Why test/why not test? Not so clear/who decides what to test for? To ease pain and suffering To save lives Not so clear/who decides what to test for? What action to take? Would you test to find out sex of child? Would you use information to choose the sex of a child? Would you test for a genetic disease that runs in your family-what would influence this decision? …..

Ethical Issues in Clinical Practice: Disclosure of the carrier status of wife to the husband (Privacy/Confidentiality) Testing of a child or adult for Huntington disease in a family with Huntington disease (Autonomy)(Psychological distress) (Human Dignity) Screening for BRCA1 / BRCA2 in general population (Cost effectiveness, Available interventions, Psychological distress) Pre Insurance/Employment screening – Denial of insurance/job (Human Rights, Fundamental freedoms) (Discrimination) Abortion of a foetus with achondroplasia, albinism, Down syndrome, Thalassemia (Rights of the unborn, Genetic burden) Prevention of marriage / having children – say for thalassemia carriers, or a ‘carrier’ of Huntington disease gene (Human Rights, Eugenics)

Prenatal screening expresses a hurtful message to those who live with disabilities. If screening leads to a decision not to bring the disabled child into existence, then the message is that a person who has a disability is unworthy of being born.

“Once we put human life in human hands, we may go down a slippery slope that knows no boundaries…”