UPIK A.

MISKAD,PhD, SpPA

Empat kelompok gangguan : 1. Terjadi mutasi ulang trinucleotide 2. Terjadi mutasi pada gen mitokondria 3. Kelainan yang berhubungan dengan pencetakan genom (Genomic Imprinting) 4. Kelainan yang berhubungan dengan Mosaicism Gonade

TABLE 5-8 Disease

-- Examples of Trinucleotide-Repeat Disorders No. of Repeats Gene FMRI (FRAXA) Locus Xq27.3 Protein FMR-1 protein (FMRP) Repeat Normal CGG 653 Disease 60200 (pre); >230 (full) 3480 (pre); >100 (full) 3480 (pre); >100 (full) 3862

EXPANSIONS AFFECTING NONCODING REGIONS Fragile-X syndrome

Friedreich ataxia

FXN

9q21.1

Frataxin

GAA

734

Myotonic dystrophy

DMPK

19q13.3

Myotonic dystrophy protein kinase (DMPK) Androgen receptor (AR) Huntingtin

CTG

537

EXPANSIONS AFFECTING CODING REGIONS Spinobulbar muscular atrophy (Kennedy disease) Huntington disease Dentatorubralpallidoluysian atrophy (Haw River syndrome) Spinocerebellar ataxia type 1 Spinocerebellar ataxia type 2 Spinocerebellar ataxia type 3 (MachadoJoseph disease) Spinocerebellar ataxia type 6 AR Xq12 CAG 936

HTT ATNL

4p16.3

CAG CAG

635 635

36121 4988

12p13.31 Atrophin-1

ATXN1 ATXN2 ATXN3

6p23 12q24.1 14q21

Ataxin-1 Ataxin-2 Ataxin-3

CAG CAG CAG

644 1531 1240

3982 3663 5584

CACNA2A

19p13.3

1A-Voltagedependent calcium channel subunit Ataxin-7

CAG

418

2133

Spinocerebellar ataxia type 7

ATXN7

3p14.1

CAG

435

37306

Ada beberapa prinsip : Penyebab mutasi yang berhubungan dengan ekspansi trinucleotide, biasanya nukcleotide G dan C. Kecenderungan ekspansi sangat bergantung pada jenis kelamin induk transmisi Pada 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 trinucleotiderepeat 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 1535 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, 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. Highthroughput 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)

2.

analisis linkage utk identifikasi haplotipes yg berhubungan dg penyakit.

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 project 2. 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.