FRAGILE X SYNDROME

Group IV: Navarro, Jonah Paul Nieto, Dianne Margareth Nonescan, Shigela Harieth Oasay, Mary-Joy Obial, Khim Ocampo, Ana Hiyas

Children and adults with fragile x syndrome are not very different in appearance form other people. In fact, when a baby is born with the condition, parent and doctors usually have no idea that anything is wrong. Often, parents and others become concerned only when a baby is delayed in early development skills such as sitting, walking or talking.

The Case

ELDER BROTHER 2 years and 9 months Was born at term Birth Weight: 3260g Gross psychomotor retardation

Physical Exam

Head control at the age of 5 months Sat alone at 12 months Walk at 20 months but without any meaningful words
Height: 94.5cm Weight: 13.2kg Head circumference: 47.8cm

Physical Appearance
Long and square shaped face Prominent forehead Absence of left lower incisor Testes were not enlarged Hyperactivity Short attention span Poor eye contact Withdrawal

Behavioral abnormalities

Psychometric test: development quotient was 38% that of a normal child of the same age Diagnostic and statistical manual of mental disorders IV: criteria for childhood autistic disorders Electroencephalography (EEG): Normal Acoustic brainstem response (ABR): Normal

YOUNGER BROTHER
1 year and 8 months Birth weight: 2786 g Psychomotor development was moderately retarded Head control at the age of 4 months Sat alone at 10 months Can stand up at 20 months but he never spoke any meaningful words

Physical examination
Normal height Normal head circumference

Physical appearance
Square-shaped face Prominent forehead Everted ears Absence of lower incisor

Behavioral abnormalities
Short attention span but no social aversion or hand mannerisms

EEG and ABR were not remarkable but massive epileptic discharges during sleep had become evident on the bilateral parietooccipital regions since 1 year and 6 months of age.

MATERNAL AUNT
Motor development almost normal but speech was grossly delaye Attended special educational schools Anticonvulsant drugs- EEG abnormalities

Physical Examination
Shyness Mentally retarded Long face with prognathism Menstruation at the age of 13 Speech disturbance was characterized by lack of fluency, echolalia and inappropriate grammatical quotient of 45 ( normal range, 77-124)

YOUNGER SISTER
Birth weight: 3462 g Normal psychomotor development except for speech Square shaped face but appeared normal Intelligence score was 85% that of a normal age

Answers to Guide Questions:

1. What clinical features do prepubertal male patients affected with Fragile X Syndrome have?

Clinical features of Prepubertal Males

Birth weight a mean at the approximately 70th percentile Height: mostly between 50th and 97th percentile Head circumference: slightly increased

Clinical features of Prepubertal Males

Developmental delay: -- Sit alone at 10 months -- Walk at 20.6 months -- First meaningful words at 20 months Abnormal behavior: -- Hyperactivity -- Hand mannerisms -- Excessive shyness -- Tantrum -- Autistic features

2. Why is it important for medical personnel and scientist to understand the molecular basis of Fragile X Syndrome?

Why is it important to understand Fragile X Syndrome?

It is considered one of the most commonly inherited causes of mental retardation.

Timely diagnosis allows children to receive early intervention services and families to receive genetic counseling (Visootsak, 2005)

Why is it important to understand Fragile X Syndrome?

Patients with Fragile X Syndrome exhibit non-specific clinical features similar to that of autistic characteristics.

This syndrome can be comparable to that of Down Syndrome.

3. What is the Sherman Paradox in Fragile X Syndrome, and how can this paradox be resolved on a molecular basis?

The Sherman Paradox

The Sherman Paradox

The Sherman Paradox refers to an anomalous pattern of inheritance found in Fragile X syndrome. The phenomenon is also referred to as anticipation or dynamic mutation.

The Sherman Paradox

Anticipation is seen in genetic diseases that are passed through generations. Causes subsequent generations in a family to be more severely affected by a disease. It does this by increasing the number of triplet repeats in the fragile area of the x chromosome through the generations. It also causes the disease to strike with a greater frequency.

FMR1 (fragile X Mental Retardation 1)

is a human gene that codes for a protein called fragile X mental retardation protein, or FMRP. This protein is normally made in many tissues, especially in the brain and testes. It may play a role in the development of synaptic connections between nerve cells in the brain, where cell-to-cell communication occurs. The connections between nerve cells can change and adapt over time in response to experience (a characteristic called synaptic plasticity). FMRP may help regulate synaptic plasticity, which is important for learning and memory.

FMR1 (fragile X Mental Retardation 1)

One region of the FMR1 gene contains a 3 base Variable Number Tandem Repeat (VNTR, or more specifically, a trinucleotide repeat). The sequence CGG is repeated a number of times. In most healthy individuals, the number of CGG repeats ranges from fewer than 10 to about 40, with the median at about 29 repeats.

Molecular Perspective of Fragile X Syndrome

Southern Blotting

can detect mutations in DNA combines the use of restriction enzymes, electrophoresis, and DNA probes patterns observed = specific restriction endonuclease + probe used to visualize the restriction fragments variants of Southern Blot: Northern - electrophoresis of mRNA followed by hybridization with a specific probe Western - electrophoresis of protein followed by detection with an antibody directed against the protein of interest

Southern Blotting
1. DNA is extracted from from cells 2. DNA is cleaved into many fragments using a restriction enzyme 3. the resulting fragments are separated on the basis of size by electrophoresis (bigger=slower smaller=faster) 4. The DNA fragments in the gel are denatured and transferred (blotted) to a nitrocellulose membrane for analysis 5. A probe is used to identify the DNA fragments of interest

Southern Blotting in Fragile X Syndrome

preferred method in diagnosis can determine the extent of CGG repeat expansion & methylation status of the CpG island small premutation of the alleles = cleavage with Pst1 + hybridization with a Pfxa3 probe

Southern Blotting in Fragile X Syndrome

Double Digestion with a methylation insensitive enzyme:

to examine methylation status and CGG repeat length BssHII or EagI

Bands observed in Fragile X using Southern Blot

5.2kb band - inactive X of female 2.8 and 2.4 kb bands - active X of female and single X of normal male

Bands observed in Fragile X using Southern Blot

males w/ premutations - slightly larger 2.8kb band (longer repeat length) males w/ full mutations - slightly larger 5.2kb band (methylated & expanded FMR-1 mutation) females w/ premutations - 3 bands of normal female pattern (unmethylated active state of 2.4 & 2.8kb and methylated inactive state of normal bands) and two additional premutation bands (sometimes combined into normal bands) females w/ full mutations - expanded CGG repeat (over methylated) smear band >7.5kb in addition of normal female bands female mosaic - methylated & unmethylated in both normal & abnormal X chromosome alleles

Polymerase Chain Reaction (PCR)

permits the synthesis of millions of copies of a specific nucleotide sequence in a few hours can be used to amplify DNA sequences from any source - bacterial, viral, plant, or animal uses DNA polymerase to repetitively amplify targeted portions of DNA exponentially amplified DNA sequence can then be analyzed by gel electrophoresis, Southern hybridization, or direct sequence determination

Polymerase Chain Reaction

PCR in Fragile X Syndrome

determines more accurate CGG repeat numbers amplifying DNA regions w/ high CG content is difficult preferential amplification at the smallest allele in females failure to amplify full mutations substitution of 7-deazad GTP use of improved primers sequencing acrylamide gels Advantages - speed, sensitive, requires minimal amounts of DNA

Prenatal Diagnosis
uses

Chorionic Villus Sampling (CVS) DNA obtained in chorionic villi is analyzed using Southern blot or PCR

Numbers of CGG repeats

male or female w/ 6-50 copies = normal Male fetuses w/ 50-230 copies = asymptomatic >230 copies = fragile X syndrome Female fetuses w/ 50-230 copies = asymptomacit >230 copies = difficult to predict hypermethylation of CpG island = not always present in DNA extracted in CVS Female carriers w/ full mutations = 50% risk of mental impairment

Genetic Diseases Associated with Dynamic Mutations

Fragile X syndrome is only one of the eight human genetic diseases wherein dynamic mutation of the trinucleotide repeat has been shown to be the cause common feature = Genetic Anticipation (disease becomes severe in succeeding generations) factor in expansion of CGG repeat = Okazaki fragment slippage

Disease

Chromosom e Location

Repeated Sequence

Sex Bias of Parent Contributing Severe Form

Maternal

Normal no. of Copies

No. of Copies Associated with the Disease

Premutation: 50-230 Full mutation: 230-2000 40-62

Change in Gene Function

Fragile X syndrome

Xq27.3

CGG

6-50

Loss

Spinobulbar muscular atrophy (Kennedy disease) Myotonic dystrophy

Xq11-12

CAG

11-31

Gain

19q13.3

CTG

Maternal

5-35

Premutation: 50-80 Full mutation: 80-2000 Premutation: 30-38 Full mutation: 37-121 43-81

mRNA stability

Huntington disease

4p16.3

CAG

Paternal

9-37

Gain

Spinocerebell ar ataxia type I Fragile XE mental regardation Dentatorubral pallidoluysian atrophy

6p22-23

CAG

Paternal (possibly) ?

25-36

Gain

Xq28

CGG

6-25

Premutation: 25-200 Full mutation: >200 49-75

Loss

12p12.13

CAG

Paternal (mainly)

7-23

Gain

MachadoJoseph

14q32.1

CAG

13-36

68-79

Gain

Carriers
A Carrier is an individual who carries an altered form of a gene which can lead to having a child or offspring in future generation with a genetic disorder.

People with a normal number of repeats

Most people have about 6 to 45 repeats in their FMR1 genes. Repeats of this size are stable in families. That is, the number of repeats does not change when passed from parent to child.

People with an intermediate number of repeats

People who have an intermediate repeat size (about 45 to 55) are not at risk for having children with FXS. However, when the gene is passed on to their children, the number of repeats can grow to a premutation size. Children who have a premutation are then at risk for having children with FXS. Therefore, a person with an intermediate size repeat is at risk for having grandchildren with FXS.

People with a premutation

A premutation repeat (about 55 to 200 repeats) can grow to a full mutation when the gene is passed from a mother to a child.

People with a full mutation

If men with FXS (over 200 repeats) have children, they do not pass on the full mutation. Rather, the full mutation shrinks back to a premutation size and their daughters will have premutations. The sons of males with FXS will not be affected, because boys get a Y chromosome from their fathers instead of an X chromosome.

Transmission
Y XFragile X Boy XY Girl XXFragile

Boy XY

Girl XXFragile

XFragile

Boy YXFragile

Girl XXFragile

Boy XY

Girl XX

6. What other human genetic diseases have been attributed to dynamic mutation of a trinucleotide repeat?

Other human genetic diseases attributed to trinucleotide repeat

Huntinton's

Chorea [CAG]

-- An inherited neurodegeneration that often occurs in the fourth or fifth generation of life -- As the size of the repeat section increases over generations, the age of onset of the disease becomes earlier

Other human genetic diseases attributed to trinucleotide repeat

Friedreich ataxia [GAA]

-- Also a neurodegenerative disorder -- Expansion messes up the regulation of protein synthesis so there is a decreased amount of protein synthesized

Therapies for fragile X syndrome

Speech and Language Behaviour Cognitive development Sensory integration Goss motor development Daily living

Speech and Language

Intervention by a speech-language pathologist (SLP) is essential Delayed speech is the first sign that something is wrong with a young child. Children with Fragile X syndrome have unique speech and language disorders:

Boys with Fragile X syndrome pragmatic (conversational skills). physical, oral-motor, attention, and behavioral characteristics. integrated approach to treatment is necessary. Girls with Fragile X syndrome good verbal skills difficulty in pragmatic speech anxiety and shyness affecting their social interactions. benefit from the services of a speechlanguage pathologist (SLP).

Their speech and language are affected by:

physical, oral-motor, attention, and behavioral characteristics

A speech and language pathologist can help with this. Rather than offering the patient individual therapy in a "speech room", the SLP will design goals that can be carried out at home, in the daycare, on the playground, and with a variety of other professionals that will be working with the affected individual.

Behavioral Therapy
Intervention for difficulties with attention, anxiety, and relationships may require planning for both medication and behavior modification. Parents and educators may need to devise behavioral plans to help children with Fragile X syndrome cope with everyday demands of home, school, and community.

Behavioral interventions
including calming techniques, modified environments and behavior therapy (ABA Applied Behavioral Analysis) supervised by a psychologist, in conjunction with medication, may be beneficial for people with Fragile X syndrome.

For early childhood and school age children clear, concrete plans, with appropriate cues (e.g., visual signals for quiet mouths) and appealing rewards (e.g., stickers which lead to prizes) are essential. Older adolescents and adults may need specific behavioral plans in vocational training, so that they can function in the work setting in the most appropriate manner. Adapting and modifying are key for people with Fragile X syndrome.

Sensory Integration and Occupational Therapy

SI therapy is designed to help the individual gradually respond more appropriately to sensory input. This involves input to all of the five senses, plus proprioceptive (the sense of one's body in space) and vestibular (the sense of gravity and motion) input.

An occupational therapist (OT) is someone who is trained to provide assessment and therapy for fine motor, self-help, and sensory integration disorders. OT may be provided in early intervention, early childhood, and school programs, in work settings, and through private agencies. OT often designs a sensory diet for an individual with Fragile X syndrome, finding the best combination and timing of various sensory inputs: arousal levels, tactile defensiveness, fine motor weaknesses, and oral motor needs.

Forming a plan
Evaluation of sensory and fine-motor strengths and needs should be completed. Evaluation

Interviews with parents and caregivers may provide the assessment team with knowledge about the childs or adults typical performance and reactions in various situations.

interview with parents and teachers observation of the affected person in various situations, some formal tests.

For examples, persons with Fragile X syndrome often have difficulty in situations that are new or are noisy, crowded, or confusing, and parents may be able to describe their childs reactions and coping strategies in such instances. Observations

Home Classroom Workplace.

The occupational therapist (OT)

settings are optimal for best performance by the child or adult with Fragile X syndrome and what alterations can be made in the environment to help that person. May also observe the individual during transitions from one activity to another and when unexpected events take place and affected individual with regard to seating (postural issues) and movement.

Symptoms Seizures Mood instability

Genetic Medications (Brand names in parentheses) Carbamazepine (Tegretol) Valproic acid or divalproex (Depakote) Lithium carbonate Gabapentin (Neurontin) Lamotrigine (Lamictal) Topiramate (Topomax), tiagabine (Gabitril), and vigabatrin (Sabril) Phenobarbital and primidone (Mysoline) Methylphenidate (Ritalin, Concerta) and dexamethamphetamine (Adderall, Dexedrine) L-acetylcarnitine Venlafaxine (Effexor) and nefazodone (Serzone) Amantadine (Symmetrel) Folic acid

Attention deficit (With or without hyperactivity)

Hyperarousal Sensory over-stimulation Clonidine (Catapres TTS patches) (Often occurs with ADD/ADHD) Guanfacine (Tenex) Aggression Intermittent explosive disorder Obsessive-compulsive disorder (Often occurs with anxiety and/or depression)

Fluoxetine (Prozac) Sertraline (Zoloft) and citalopram (Celexa) Paroxetine (Paxil) Fluvoxamine (Luvox) Risperidone (Risperdal) Quetiapine (Seroquel) Olanzepine (Zyprexa)

Sleep disturbances Trazadone Melatonin

6. What other human genetic diseases have been attributed to dynamic mutation of a trinucleotide repeat?

Other human genetic diseases attributed to trinucleotide repeat

Huntinton's

Chorea [CAG]

-- An inherited neurodegeneration that often occurs in the fourth or fifth generation of life -- As the size of the repeat section increases over generations, the age of onset of the disease becomes earlier

Other human genetic diseases attributed to trinucleotide repeat

Friedreich ataxia [GAA]

-- Also a neurodegenerative disorder -- Expansion messes up the regulation of protein synthesis so there is a decreased amount of protein synthesized