Genetics of Neurological Disorders 张咸宁 zhangxianning@zju.edu.cn Tel：13105819271; 88208367 Office: A705, Research Building 2013/11

Learning Objectives 1.To become familiar with the clinical phenotypes of the major triplet repeat disorders such as fragile X syndrome, Huntington disease, myotonic dystrophy and Friedreich ataxia. 2. To understand the unique and unusual molecular mechanisms and modes of inheritance underlying these disorders. 3. To appreciate how this knowledge is translated to clinical molecular genetic testing of these disorders for purposes of diagnosis, predictive testing, and prenatal testing. 4. To become aware of some of the difficult ethical issues associated with molecular testing for these disorders.

Required Reading Thompson &Thompson Genetics in Medicine, 7th Ed （双语版，2009） pp. 302-305, Chapter 12, The Molecular and Biochemical Basis of Genetic Disease - Diseases due to the Expansion of Unstable Repeat Sequences: Biochemical and Cellular Mechanisms Case Study, 15. Fragile X Syndrome 22. Huntington Disease

在已发现的单基因遗传病（www. omim. org）中，半数以上累及神经系统。国内神经系统单基因遗传病的发病率约为109 神经系统遗传病可在任何年龄发病。出生后：半乳糖血症等，婴儿期：SMA1、Tay-Sachs病等，儿童期：DMD等，少年期：肝豆状核变性、SMA2等，青年期：腓骨肌萎缩症等，成年期：强直性肌营养不良等，成年后期：Huntington舞蹈症等，老年期：Alzheimer病等。多数神经系统遗传病在30岁之前出现症状。 神经系统遗传病病种繁多，致残和致畸率很高，治疗困难。

ALS is a lethal, paralyzing disorder of motor neurons in the brain, brainstem and spinal cord. Its onset, typically in the 6th decade of life, is age-dependent; mean onset is 55 yr, and mean survival is 3–5 yr. About 5–10% of cases are transmitted as an autosomal dominant trait familial ALS.

Classification of Neurogenetic Disorders by Presentation Dementias Muscular dystrophies and atrophies Ataxias Mental retardation/dysmorphism

Classification by Molecular Defect Point mutations Large deletions Trinucleotide repeat expansions Mitochondrial DNA mutations

Trinucleotide repeat expansions are dynamic mutations An unstable expanded repeat that changes size between parent and child.

Contrasting Features of Triplet Repeat Disorders Huntington disease AD CAG Coding region (exon 1) Fragile X syndrome X-linked CGG 5’-untranslated region Myotonic dystrophy CTG 3’-untranslated region Friedreich ataxia AR GAA Intron

Unusual Aspects of Inheritance of Trinucleotide Repeat Disorders Anticipation: tendency toward earlier age of onset and/or greater severity in each subsequent generation, due to progressive expansion of the repeat length. Parent-of-origin effects Skewed X-inactivation Methylation effects Incomplete penetrance Variable expressivity Premutation alleles: asymptomatic, but unstable, with a tendency to expand in the next generation

(A) In fragile X syndrome, the expanded repeat in the 5’ UTR of the gene triggers methylation of the promoter and prevents transcription. (B) In myotonic dystrophy, the expanded repeat in the 3’ UTR causes the mRNA transcript to sequester splicing factors in the cell nucleus, preventing the correct splicing of several unrelated genes. (C) In Huntington disease, the gene containing the expanded repeat is transcribed and translated as normal, but the protein product has an expanded polyglutamine tract that renders it toxic.

Huntington Disease (Chorea) a progressive loss of motor control, dementia, and psychiatric disorders. The brain area most noticeably damaged is the corpus striatum. The suicide rate among HD patients is >5~10 in the general population. approximately 1 in 20,000 persons of European descent. usually manifests between the ages of 30 and 50 years, although it has been observed as early as 1 year of age and as late as 80 years of age.

Woody Guthrie (1912-1967)

HD gene----Hero! In 1983, HD was the first genetic disease to be localized to a chromosome location (4p16.3) with RFLP linkage analysis. (Gusella et al.) Robertson:“The beginning of the end of dilemma?” (Nature) The HD gene, Huntingtin, was isolated in 1993 after a decade of intense collaborative efforts among many laboratories from various countries and officially designated HD. (Gusella et al.) Little:“Huntington’s disease: The end of the beginning”(Nature)

Trinucleotide CAG repeat sizes in HD gene (huntingtin) Normal ≤26 Mutable 27-35 Reduced penetrance 36-39 Fully penetrance ≥40

Clinical Case Mary (35 y.o.), Samuel (30 y.o.), and Alice (29 y.o.) are siblings at 50% risk to inherit Huntington disease from their father, John, who was found to have a mutable normal allele when he was tested following diagnosis of his brother, Bart. All three siblings chose molecular genetic testing following genetic counseling and neurologic evaluation. All have normal neurologic examinations.

What do these results mean? John Mutable normal Bart Mary 38 CAG repeats Alice 42 CAG repeats Samuel 35 CAG repeats What do these results mean?

Diagnosis Molecular Genetic Testing

Samuel (35 repeats) is told that he has a mutable normal allele Samuel (35 repeats) is told that he has a mutable normal allele. Expansions of 27-35 CAG repeats have never been associated with clinical symptoms of HD; however, his children are at some risk to inherit an allele with a larger allele size which could result in symptomatic HD. Mary Alice Samuel (30 y.o.) 35 CAG repeats Mutable normal allele

Mary (38 repeats) is told that she has a reduced penetrance allele Mary (38 repeats) is told that she has a reduced penetrance allele. Expansions of 36-40 CAG repeats may or may not cause symptoms of HD during a normal life span. The onset of symptoms may be later than typically observed. Mary's children are at 50% risk for inheriting the abnormal allele, which could remain in the reduced penetrance range or expand into the full penetrance range. Mary (35 y.o.) 38 CAG repeats Reduced penetrance allele Alice Samuel

Alice (42 repeats) is told that she has a full penetrance allele Alice (42 repeats) is told that she has a full penetrance allele.. Expansions of 41 CAG repeats or greater are always associated with symptomatic HD if the individual lives a normal life span. Alice's children are at 50% risk to inherit the full penetrance allele and therefore to develop HD. Alice (29 y.o.) 42 CAG repeats Full penetrance allele Mary Samuel

Anticipation the phenomenon in which increasing disease severity or decreasing age of onset is observed in successive generations, is known to occur in HD. occurs more commonly in paternal transmission of the mutated allele. The phenomenon of anticipation arises from instability of the CAG repeat during spermatogenesis. Large expansions (i.e., an increase in allele size >27 CAG repeats) occur almost exclusively through paternal transmission. Most often children with juvenile-onset disease have inherited the expanded allele from their fathers.

Predominant Clinical Features of Fragile X Syndrome in Males Prepubertal Delayed developmental milestones: Sit alone, 10 mo; Walk, 20.6 mo; First clear words, 20 mo. Developmental delay Abnormal behavior: Tantrums; Hyperactivity; Autism Mental retardation: IQ 30 to 50 Abnormal craniofacies: Long face; Prominent forehead; Large ears; Prominent jaw Postpubertal MR; Pronounced; Craniofacies; Macroorchidism Additional Features Strabismus; Joint hyperextensibility; Mitral valve prolapse; Soft, smooth skin

A: Two-yr-old male with a full mutation exhibiting a relatively normal appearance with an elongated face and prominent ears; also note tapering fingers, a minor anomaly. B: At age 5 years, his head is large with large ears and a prominent jaw. C: At age 22 years.

CGG Repeat in Fragile X Syndrome Normal range: 6-54 Premutation range: 52-200 Full mutation range: 200- >1000 Alleles with >200 repeats are hypermethylated, transcriptionally repressed

Risk of Expansion of Fragile X Premutations Length of maternal premutation Incidence of full mutation in offspring 56-59 13% 60-69 20% 70-79 58% 80-89 73% 90-99 94% 100-109 100% 120-129 Nolin et al., Am J Hum Genet 1996

Is the fragile X premutation really asymptomatic? Recent reports of premature ovarian failure in female premutation carriers Late-onset tremor-ataxia-dementia syndrome in male premutation carriers May be due to mRNA interference with expression of the normal FMR1 allele or of other genes

A New Fragile X Testing Dilemma Genetic counselors and obstetricians are beginning to order fragile X carrier screening on all pregnant women, regardless of family history Unexpected premutation alleles are being identified, leading to amniocentesis Pregnancies in which the fetus is found to have only the premutation are typically continued The resulting child is now labeled with a late-onset genetic disease for which there is no treatment

NIH Task Force on Genetic Testing

Myotonic Dystrophy AD Myotonia, muscular dystrophy Cataracts, hypogonadism, frontal balding Severe neonatal form due to dramatic CTG repeat expansion from affected mother only

A three-generation family affected with myotonic dystrophy A three-generation family affected with myotonic dystrophy. The degree of severity increases in each generation. The grandmother (right) is only slightly affected, but the mother (left) has a characteristic narrow face and somewhat limited facial expression. The baby is more severely affected and has the facial features of children with neonatal-onset myotonic dystrophy, including an open, triangle-shaped mouth. The infant has more than 1000 copies of the trinucleotide repeat, whereas the mother and grandmother each have approximately 100 repeats.

Friedreich Ataxia AR The most common of the hereditary ataxias in the Caucasian population. Gait disturbance in childhood Upper extremity ataxia Absent reflexes Intellectual decline Progressive cardiomyopathy Small proportion due to point mutations rather than GAA repeat expansion

1 2 3 Friedreich Ataxia PCR Normal allele size

Acknowledge（PPT特别鸣谢！） UCLA David Geffen School of Medicine www.medsch.ucla.edu/ANGEL/ Prof. Grody WW (Divisions of Medical Genetics and Molecular Pathology), et al.