UPIK A. MISKAD,PhD, SpPA

Empat kelompok gangguan :1.Terjadi mutasi ulang trinucleotide2.Terjadi mutasi pada gen mitokondria3.Kelainan yang berhubungan dengan

pencetakan genom (Genomic Imprinting)4.Kelainan yang berhubungan dengan

‘Mosaicism Gonade’

TABLE 5-8 -- Examples of Trinucleotide-Repeat Disorders No. of Repeats

Disease Gene Locus Protein Repeat Normal Disease EXPANSIONS AFFECTING NONCODING REGIONS

Fragile-X syndrome FMRI (FRAXA)

Xq27.3 FMR-1 protein (FMRP)

CGG 6–53 60–200 (pre); >230 (full)

Friedreich ataxia FXN 9q21.1 Frataxin GAA 7–34 34–80 (pre); >100 (full)

Myotonic dystrophy DMPK 19q13.3 Myotonic dystrophy protein kinase (DMPK)

CTG 5–37 34–80 (pre); >100 (full)

EXPANSIONS AFFECTING CODING REGIONS

Spinobulbar muscular atrophy (Kennedy disease)

AR Xq12 Androgen receptor (AR)

CAG 9–36 38–62

Huntington disease HTT 4p16.3 Huntingtin CAG 6–35 36–121

Dentatorubral-pallidoluysian atrophy (Haw River syndrome)

ATNL 12p13.31 Atrophin-1 CAG 6–35 49–88

Spinocerebellar ataxia type 1

ATXN1 6p23 Ataxin-1 CAG 6–44 39–82

Spinocerebellar ataxia type 2

ATXN2 12q24.1 Ataxin-2 CAG 15–31 36–63

Spinocerebellar ataxia type 3 (Machado-Joseph disease)

ATXN3 14q21 Ataxin-3 CAG 12–40 55–84

Spinocerebellar ataxia type 6

CACNA2A 19p13.3 α1A-Voltage-dependent calcium channel subunit

CAG 4–18 21–33

Spinocerebellar ataxia type 7

ATXN7 3p14.1 Ataxin-7 CAG 4–35 37–306

Ada beberapa prinsip : Penyebab mutasi yang berhubungan dengan

ekspansi trinucleotide, biasanya nukcleotide G dan C.

Kecenderungan ekspansi sangat bergantung pada jenis kelamin induk transmisiPada Fragile-X Syndrome, ekspansi terjadi selama oogenesis, sedangkan pd Huntington Disease terjadi selama spermatogenesis.

Dari sudut pandang mekanisme, mutasi dapat dibagi menjadi 2 kelompok :◦ Fragile-X Syndrome & Myotonic Distrofi ekspansi

berulang terjadi pada regio non-coding◦ Huntington Disease ekspansi terjadi pada regio coding

Figure 5-24 Sites of expansion and the affected sequence in selected diseases caused by nucleotide-repeat mutations. UTR, untranslated region.*Though not strictly a trinucleotide-repeat disease, progressive myoclonus epilepsy is caused, like others in this group, by a heritable DNA expansion. The expanded segment is in the promoter region of the gene.

Figure 5-25 Fragile X, seen as discontinuity of staining. (Courtesy of Dr. Patricia Howard-Peebles, University of Texas Southwestern Medical Center, Dallas, TX.)

Merupakan prototype penyakit yang mutasinya ditandai oleh urutan 3 nukleotida yang berulang & panjang (nukleotida guanin (G) & cytosine (C).

Kebanyakan terjadi pd laki-laki. Retardasi mental (> laki2), wajah panjang

dgn mandibula besar, telinga besar, macroorchidism), sendi hiperekstensi, palatum tinggi, & prolaps katup mitral

Mutasi pada gen FMR1 yang terletak di Xq27.3

Figure 5-26 Fragile-X pedigree. Note that in the first generation all sons are normal and all females are carriers. During oogenesis in the carrier female, premutation expands to full mutation; hence,

in the next generation all males who inherit the X with full mutation are affected. However, only 50% of females who inherit the full mutation are affected, and only mildly. (Courtesy of Dr. Nancy Schneider, Department of Pathology, University of Texas Southwestern Medical Center, Dallas,

TX.)

Figure 5-27 A model for the action of familial mental retardation protein (FMRP) in neurons. (Adapted from Hin P, Warren ST: New insights into fragile X syndrome:

from molecules to neurobehavior. Trends Biochem Sci 28:152, 2003.)

LEBER HEREDITARY OPTIC NEUROPATHY Pewarisan mtDNA berbeda dengan pewarisan

DNA inti sel Berkaitan dengan pewarisan maternal Ibu mewariskan gen mitokondria kpd semua

keturunannya, tetapi anak perempuan saja yang mewariskan mtDNA kpd keturunannya.

Jarang ditemukan Penurunan penglihatan pertama pd umur 15-

35 thn kebutaan

Figure 5-28 Pedigree of Leber hereditary optic neuropathy, a disorder caused by mutation in mitochondrial DNA. Note that all progeny of an affected male (shaded squares) are normal, but all children, male and female, of the affected female (shaded circles) manifest disease.

Manusia mewarisi 2 salinan dr setiap gen, yg tdpt di kromosom homolog ibu & ayah.

Ada perbedaa fungsional antara gen ibu & gen ayah

Imprinting terjadi di ovum/sperma, & diwariskan ke semua sel somatik yg berasal dr zigot.

Contoh : Prader-Willi Syndrom & Angelman Syndrom

Gejala : RM, tubuh pendek, hipotonia, kegemukan, tangan & kaki kecil, hipogonadisme.

Delesi interstitial band del(15)(q11.2q13) Pada semua kasus, delesi mengenai

kromosom 15 yg berasal dr ayah

Gejala : RM, ataksia, kejang, & “happy puppet syndrome”

Delesi pd 15q12 maternal Gen yg berperngaruh : ligase ubiquitin

(UBE3A).

Figure 5-29 Diagrammatic representation of Prader-Willi and Angelman syndromes.

Akibat mutasi postzigot pd perkembangan awal embrio.

Fenotipik dr orgtua N yg telah germ line mosaicism → mutasi penybb penykt kpd keturunan mlalui gamet mutan.

Progenital cells yg dr gamet pembawa mutasi → kemungkinan >1 anak akan spt orgtua yg t`pengaruh

Deteksi molekuler penyakit herediter memiliki

bbrp keunggulan:- Tes molekular yg sgt sensitif Cth: penggunaan amplifikasi PCR →

multiplikasi DNA atau RNA jutaan kali shg ckp diperlukan 1 atau 100 sel utk analisis

- Dasar tes DNA tdk b`gtg pd produk gen yg mgkn di produksi hanya sel khusus ttt (otak) atau ekspresi suatu gen mgkn br tjd pd usia lanjut

Analisis Prenatal Analisis Postnatal

Analisis genetik prenatal → ditawarkan pd semua pasien yg beresiko memiliki keturunan cytogenetically abnormal

INDIKASI ANALISIS GENETIK PRENATAL :- Ibu lanjut usia (>35 th) → > Trisomi- Orgtua pembawa translokasi yg seimbang,

translokasi Robertsonian atau Inversi- Org tua dg anak sblmnya kelainan

kromosom.- Janin yg t`deteksi dg kelainan USG.- Org tua yg merupakan pembawa gangguan

genetik X-linked (utk menentukan jenis kelamin janin).

- Kelainan tingkat AFP, β HCG, Estriol dilakukan sbg Triple Test

INDIKASI GENETIK POSTNATAL :- Bbrp anomali kongenital- Retardasi mental- Suspek aneuploidy (Down Syndrom)- Suspek ketidakseimbangan autosom

(Prader – Willi Syndrome)- Suspek Fragile – X Sindrome- Infertilitas (utk menyingkirkan kelainan

kromosom sex)- Bbrp abortus spontan

1. Diagnosis & pengelolaan cancer:◦ Deteksi pd tumor – mutasi spesifik yg di dpt &

perubahan sitogenetik (BCR – ABL1 pd CML)◦ Penentuan clonality sbg indikator kondisi

neoplastik◦ Identifikasi perubahan genetik spesifik (HER2

Neu/ ERBB2) pd Ca Mammae atau EGFR pd Ca Paru

◦ Penentuan efikasi pengobatan (minimal sisa deteksi penyakit pd BCR – ABL1 oleh PCR pd CML)

◦ Deteksi pd Gleevec – btk resisten CML & tumor stroma gastrointestinal

2. Diagnosis & penatalaksanaan penyakit menular

- Deteksi mikroorganisme – bhn genetik spesifik utk diagnosis definitif (HIV, mikrobakteria, human papilomavirus, virus herpes dlm SSP)

- Identifikasi perubahan genetik spesifik dl genom mikroba yg b`kaitan dg resistensi obat

- Penentuan efikasi pengobatan (penyakit virus dlm HIV & infeksi virus Hepatitis C)

Analisis PCR → melibatkan amplifikasi DNA scr eksponensial dan memerlukan jumlah bhn awal yang sangat sedikit.

DIRECT DETECTION OF DNA SEQUENCE ALTERATIONS BY DNA SEQUENCING

- DNA dpt di urutkan utk m`dptkan bacaan dr urutan nukleotida & perbandingan dg urutan N (wild type) → mutasi dpt di identifikasi-Bbrp kelainan dg warisan resesif dikaitkan dg jumlah t`batas mutasi berulang, spt Cystic Fibrosis-Org dg pewarisan dominan dpt memiliki mutasi diseluruh wilayah pengkodean -Menggunakan ‘gen chip’ (microarrays) → urutan gen atau bagian dr gen

Figure 5-30 Microarray-based DNA sequencing. A, A low-power digitized scan of a "gene chip" that is no larger than a nickel in size but is capable of sequencing thousands of base pairs of DNA. High-

throughput microarrays have been used for sequencing whole organisms (such as viruses), organelles (such as mitochondria), and entire human chromosomes. B, A high-resolution view of the gene chip

illustrates hybridization patterns corresponding to a stretch of DNA sequence. Typically, a computerized algorithm is available that can convert the individual hybridization patterns across the

entire chip into actual sequence data within a matter of minutes ("conventional" sequencing technologies would require days to weeks for such analysis). Here, the upper sequence is the reference

(wild-type) sequence, while the lower one corresponds to the test sample sequence. As shown, the computerized algorithm has identified a C→G mutation in the test sample.

Enzym yg mengenali & memotong DNA mjd urutan ttt

Allele specific extension utk mengidentifikasi mutasi DNA meskipun dlm heterogen sel N dan abnormal

Teknologi dasar m`gunakan indikator fluorophore → mendeteksi ada/tdk ada mutasi dlm “real time”

Analisis PCR & analisis Southern blot → mutasi yg m`pengaruhi panjang DNA (delesi/ekspansi)

Figure 5-31 Allele-specific PCR for mutation detection in a heterogeneous sample containing an admixture of normal and mutant DNA. Nucleotides complementary to the mutant and wild-type nucleotides at the queried base position are labeled with different fluorophores, such that incorporation into the resulting PCR product yields fluorescent

signals of varying intensity based on the ratio of mutant to wild-type DNA present.

Figure 5-32 Diagnostic application of PCR and Southern blot analysis in fragile-X syndrome. With PCR the differences in the size of CGG repeats between normal and premutation give rise to products of different sizes and mobility. With a full mutation, the region between the primers is too large to be

amplified by conventional PCR. In Southern blot analysis the DNA is cut by enzymes that flank the CGG repeat region, and is then probed with a complementary DNA that binds to the affected part of the gene. A single small band is seen in normal males, a band of higher molecular weight in males with premutation,

and a very large (usually diffuse) band in those with the full mutation.

Terdiri atas 2 jenis:1.SNPs (Single Nucleotide Polymorphisms)

◦ analisis linkage utk identifikasi haplotipes yg berhubungan dg penyakit.

2.Repeat-Length Polymorphism◦ Mikrosatelite : < 1 kilobase & ditandai dg

pengulangan 2 – 6 psg basa◦ Minisatelite : > 1 – 3 kilobase & ditandai

pengulangan 15 – 70 psg basa

Figure 5-33 DNA polymorphisms resulting from a variable number of CA repeats. The three alleles produce PCR products of different sizes, thus identifying their origins from specific

chromosomes. In the example depicted, allele C is linked to a mutation responsible for autosomal-dominant polycystic kidney disease (PKD). Application of this to detect progeny carrying the disease-related gene (red symbols) is illustrated in one hypothetical pedigree.

Males (squares); females (circles).

GWAS (Genome Wide Assosiation Studies) Utk identifikasi varian genetika yg berkaitan

dg pe↑ resiko penyakit ttt Pengaktifan GWAS ada 2 :

1. Hap map project2. High-density SMP-chip technology

Figure 5-34 General scheme for conducting a genome wide association study (GWAS). Using the publicly available "HapMap" data, the human genome is divided into "haplotypes" or regions of contiguous DNA inherited as a block, each identified by one or a few "tag" SNPs that identify the haplotype. In the example shown,

locus 1 contains three haplotypes defined by different combinations of SNPs, where white signifies the more common "normal" sequence and each color designates a different SNP; thus, these haplotypes can be distinguished by assaying for only the blue and purple "tag" SNPs. Thereafter, high density SNP chips

are constructed that contain these "tag" SNPs, in order to enable an unbiased genome-wide assessment of shared haplotypes between disease and control populations. Of note, "disease" refers to any defined phenotype, and could pertain to an actual disease entity like hypertension, or simply a quantitative trait like hair or eye color. Next, DNA obtained from the two cohorts is analyzed for overrepresented SNPs in the disease population ("cases") versus the control samples-

this is known as a case-control study. The most significant shared genomic regions of interest are then examined for candidate genes of interest-an example shown here in a search for loci associated with hypertension is angiotensinogen, a gene on chromosome 1 whose product regulates vascular smooth muscle tone. The final step is to perform a second case control study, this time using SNPs located within the gene of interest in order to confirm or refute the association with the trait, often in an independent population from the one in which the initial GWAS was conducted. In this example, individual SNPs within angiotensinogen gene

are denoted as red vertical bars, and these SNPs will be tested in the second round of case-control study.

Southern Blotting Fluorescence in Situ Hybridization (FISH) Array-Based Comparative Genomic

Hybridization (Array CGH)

Figure 5-35 FISH studies using multicolor FISH in a child with an undetermined abnormality. This technique uses ratio-labeled probes labeled with 23 distinct mixtures of 5 fluorophores to create a unique "color" for each chromosome. This analysis revealed a derivative chromosome 9, with 9p

containing additional material from 22q. (Courtesy of Dr. Stuart Schwartz, Department of Pathology, University of Chicago, Chicago, IL.)

Figure 5-36 A, Array CGH is performed by hybridization of fluorescently labeled "test" DNA and "control" DNA on a slide that contains thousands of probes corresponding to defined chromosomal regions across the human genome. The resolution of most currently available array CGH assays is in the order of

about 200 to 500 kilobases. Higher power view of the array demonstrates copy number aberrations in the "test" sample (Cy5, red), including regions of amplification (spots with excess of red signal) and deletion (spots with excess of green signal); yellow spots correspond to regions of normal (diploid) copy number. B, The hybridization signals are digitized, resulting in a virtual karyotype of the genome of the "test" sample. In the illustrated example, array CGH of a cancer cell line identifies an amplification on the distal long arm of chromosome 8, which corresponds to increased number of the oncogenic MYC. (A, From Snijders AM et al.: Assembly of microarrays for genome-wide measurement of DNA copy number. Nat Genet 29:263, 2001. Web Figure A, Copyright

2001. Reprinted by permission from Macmillan Publishers Ltd.)

Merupakan modifikasi kimia dr DNA atau yg diwariskan yg tidak mengubah urutan DNA itu sendiri.

Perubahan meliputi metilasi DNA & asetilasi dari histon.

Ekspresi gen sering berhubungan dgn tingkat metilasi DNA, biasanya cytosines, khususnya pada CG.

Merupakan prinsip untuk meggunakan analisis ekspresi mRNA dlm diagnosis penyakit genetik.

Diagnosis berdasarkan DNA lebih stabil bila dibandingkan dgn analisis RNA.

Analisis RNA sangat penting dlm pemeriksaan deteksi & kuantifikasi virus RNA.