제 3 장 DNA 분석

ㆍ연구 및 유전상담을 위해 DNA 분석 이용

ㆍDNA 분석의 임상적 적용법은 간접적 유전자추적과 직접적 유전자 돌연변이 분석으로 나눌 수 있다.

ㆍ문제의 가계의 중요 멤버의 DNA를 분리하여 사용

1. 간접적 돌연변이 유전자 추적

ㆍ질병 유전자가 동정되지 않은 경우에 질병 유전자와 인접한 marker를 추적하여 가족내 질병인자의 흐름을 추적

Figure 3.1 Examples of restriction emzymes and their recognition sites

ㆍ가족내 돌연변이 추적을 위해 DNA의 다형성(polymorphism)을 이용

Table 3.1 DNA polymorphism

Polymorphism

Synonym(s)

polymorphic allele

Definition(s)

Natural variations in a gene, DNA sequence, or chromosome that have no adverse effects on the individual and occur with fairly high frequency in the general population

Definition from: GeneTests from the University of Washington and the National Center for Biotechnology Information

Polymorphism involves one of two or more variants of a particular DNA sequence. The most common type of polymorphism involves variation at a single base pair. Polymorphisms can also be much larger in size and involve long stretches of DNA. Called a single nucleotide polymorphism, or SNP (pronounced "snip"), scientists are studying how SNPs in the human genome correlate with disease, drug response, and other phenotypes.

Definition from: Talking Glossary of Genetic Terms from the National Human Genome Research Institute

Difference in DNA sequence among individuals. Genetic variations occurring in more than 1% of a population would be considered useful polymorphisms for genetic linkage analysis.

출처: http://ghr.nlm.nih.gov/glossary=polymorphism

ㆍ특정 부위의 DNA의 다형성은 다형성의 형태가 다양하고 그 빈도가 높아야 유용하다.

-주로 사용되는  다형성에는 자리 다형성과 길이 다형성이 있다. 자리 다형성의 예는 RFLP를 들 수 있고, 길이 다형성에는 microsatellite와 minisatellite가 있다.

ㆍ염기배열의 다형성은 비 암호부위에 존재하거나 silent mutation으로 인하여 임상적으로 아무런 이상이 없다.

Silent mutation.

A silent base-pair substitution alters a codon but does not result in a change in the amino acid.

In this case both GGC and GGU code for Glycine.

출처: http://bio1151.nicerweb.com/Locked/media/ch17/mutation_silent.html

ㆍ이러한 염기서열 다형성의 6분의 1은 제한효소로 확인할 수 있다.

ㆍ제한효소 자리 다형성(RFLP)의 경우 시료 DNA를 제한효소로 소화한 다음 전기영동으로 분리하고, 특이적인 DNA probe(해당 다형성부위를 포함하는 DNA 절편을 표지한 단선 DNA)를 이용하여, 전기영동해서 분리된 DNA 조각의 크기(위치)를 알아냄으로서 다형성을 나타내는 DNA 시료를 비교할 수 있다. southern blot analysis(Fig. 3.8)라고 하는 방법으로 RFLP를 분석할 수 있다(Fig. 3.2, Fig. 3.3).

Figure 3.2 Factor IX intragenic RFLP

Restriction Fragment Length Polymorphism (RFLP)

Introduction

Restriction Fragment Length Polymorphism (RFLP) is a difference in homologous DNA sequences that can be detected by the presence of fragments of different lengths after digestion of the DNA samples in question with specific restriction endonucleases. RFLP, as a molecular marker, is specific to a single clone/restriction enzyme combination.

Most RFLP markers are co-dominant (both alleles in heterozygous sample will be detected) and highly locus-specific.

An RFLP probe is a labeled DNA sequence that hybridizes with one or more fragments of the digested DNA sample after they were separated by gel electrophoresis, thus revealing a unique blotting pattern characteristic to a specific genotype at a specific locus. Short, single- or low-copy genomic DNA or cDNA clones are typically used as RFLP probes.

The RFLP probes are frequently used in genome mapping and in variation analysis (genotyping, forensics, paternity tests, hereditary disease diagnostics, etc.).

How It Works

SNPs or INDELs can create or abolish restriction endonuclease (RE) recognition sites, thus affecting quantities and length of DNA fragments resulting from RE digestion.

Genotyping

Developing RFLP probes

Total DNA is digested with a methylation-sensitive enzyme (for example, PstI), thereby enriching the library for single- or low-copy expressed sequences (PstI clones are based on the suggestion that expressed genes are not methylated).

The digested DNA is size-fractionated on a preparative agarose gel, and fragments ranging from 500 to 2000 bp are excised, eluted and cloned into a plasmid vector (for example, pUC18).

Digests of the plasmids are screened to check for inserts.

Southern blots of the inserts can be probed with total sheared DNA to select clones that hybridize to single- and low-copy sequences.

The probes are screened for RFLPs using genomic DNA of different genotypes digested with restriction endonucleases. Typically, in species with moderate to high polymorphism rates, two to four restriction endonucleases are used such as EcoRI, EcoRV, and HindIII. In species with low polymorphism rates, additional restriction endonucleases can be tested to increase the chance of finding polymorphism.

PCR-RFLP

Isolation of sufficient DNA for RFLP analysis is time consuming and labor intensive. However, PCR can be used to amplify very small amounts of DNA, usually in 2-3 hours, to the levels required for RFLP analysis. Therefore, more samples can be analyzed in a shorter time. An alternative name for the technique is Cleaved Amplified Polymorphic Sequence (CAPS) assay.

출처: http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml

※Fig. 3.3에 대해서 설명해 봅시다.

ㆍ자리다형성은 두가지 형태로만 존재하므로 만약, 가족 중 유전자 추적에

Figure 3.3 Pedigree and DNA analysis

Figure 3.4 AC microsatellite polymorphism

중요한 위치에 있는 사람들이 homozygous 한 다형성을 갖고 있으면, 그 다형성은 유전자 추적에 사용할 수 가 없을 것이다.

ㆍ이러한 단점은 heterozygous한 빈도가 높은 길이 다형성에 의해 해결할 수 있다.

ㆍ길이다형성은 microsatellite와 minisatellite를 이용하며, microsatellite의 경우 반복서열의 70%에 있어서 그 반복횟수가 다르므로 유용하게 사용될 수 있다(Fig. 3.5).

Autosomal dominant polycystic kidney

Autosomal dominant polycystic kidney disease ("ADPKD", "autosomal dominant PKD" or "Adult-onset PKD") is an inherited systemic disorder that predominantly affects the kidneys, but may affect other organs including the liver, pancreas, brain, and arterial blood vessels. Approximately 50% of people with this disease will develop end stage kidney disease and require dialysis or kidney transplantation. Progression to end stage kidney disease usually happens in the 4th to 6th decades of life.[1] Autosomal dominant polycystic kidney disease occurs worldwide and affects about 1 in 400 to 1 in 1000 people.[2][3]

Defects in two genes are thought to be responsible for ADPKD. In 85% of patients, ADPKD is caused by mutations in the gene PKD1 on chromosome 16 (TRPP1); in 15% of patients mutations in PKD2 (TRPP2) are causative.[1]

Autosomal recessive polycystic kidney disease is a distinct disease that also leads to cysts in the kidneys and liver, typically presents in childhood, only affects about 1 in 20,000 people[4] and has different causes and prognosis.

Contents

1 Pathophysiology

2 Genetics

2.1 PKD and the "two hit" hypothesis

2.2 Relation to other rare genetic disorders

3 Diagnosis

4 Treatment

5 Prognosis

6 References

7 Additional images

8 External links

Pathophysiology

Recent studies in fundamental cell biology of cilia and flagella using experimental model organisms such as the round worm Caenorhabditis elegans and the mouse Mus musculus have shed light on how PKD develops in human patients.[5]

All cilia and flagella are constructed and maintained, by the process of intraflagellar transport, a cellular function that is also essential for the insertion of proteins at specific sites along cilia and flagella membranes. These inserted membrane proteins can initiate environmental sensing and intracellular signaling pathways. They play a special role in the cilia of renal epithelial cells, and are thought to be critical for normal renal cell development and function and are sorted out and localized to the cilia of renal epithelial cells by the aforementioned intraflagellar transport mechanism. Ciliated epithelial cells line the lumen of the urinary collecting ducts and sense the flow of urine. Failure in flow-sensing signaling results in programmed cell death (apoptosis) of these renal epithelial cells, producing the characteristic multiple cysts of PKD. PKD may result from mutations of signaling and environmental sensing proteins, or failure in intraflagellar transport.[6]

Illustration of PKD1 and PKD2 proteins at the cell membrane.

Two PKD genes, PKD1 and PKD2, encode membrane proteins that localize to a non-motile cilium on the renal tube cell. Polycystin-2 encoded by PKD2 gene is a calcium channel that allows extracellular calcium ions to enter the cell. Polycystin-1, encoded by PKD1 gene, is thought to be associated with polycystin-2 protein and regulates polycystin-2's channel activity. The calcium ions are important cellular messengers, which trigger complicated biochemical pathways that lead to quiescence and differentiation. Malfunctions of polycystin-1 or polycystin-2 proteins, defects in the assembly of the cilium on the renal tube cell, failures in targeting these two proteins to the cilium, and deregulations of calcium signaling all likely cause the occurrence of PKD.

Genetics

As stated above, defects in two genes are thought to be responsible for ADPKD. In 85% of patients, ADPKD is caused by mutations in the gene PKD1 on chromosome 16 (TRPP1); in 15% of patients mutations in PKD2 (TRPP2) are causative.[1][7]

PKD and the "two hit" hypothesis

The two hit hypothesis (aka Knudson hypothesis ) is often used to explain the manifestation of polycystic kidney disease later in life even though the mutation is present at birth. This term is borrowed from cancer research stating that both copies of the gene present in the genome have to be "silenced" before cancer manifests itself (in Knudson's case the silenced gene was Rb1). In ADPKD the original "hit" is congenital (in either the PKD1 or PKD2 genes) and the subsequent "hit" occurs later in life as the cells grow and divide. The two hit hypothesis as it relates to PKD was originally proposed by Reeders in 1992.[8] Support for this hypothesis comes from the fact that ARPKD patients develop disease at birth, and somatic mutations in the "normal" copy of PKD1 or PKD2 have been found in cyst-lining epithelia

Relation to other rare genetic disorders

Recent findings in genetic research have suggested that a large number of genetic disorders, both genetic syndromes and genetic diseases, that were not previously identified in the medical literature as related, may be, in fact, highly related in the genetypical root cause of the widely-varying, phenotypically-observed disorders. Thus, PKD is a ciliopathy. Other known ciliopathies include primary ciliary dyskinesia, Bardet-Biedl syndrome, polycystic liver disease, nephronophthisis, Alstrom syndrome, Meckel-Gruber syndrome, and some forms of retinal degeneration.[9]

Diagnosis

Abdominal CT scan of an adult with autosomal dominant polycystic kidney disease. Extensive cyst formation is seen over both kidneys, with a few cysts in the liver as well. Coronal plane.

A definite diagnosis of ADPKD relies on imaging or molecular genetic testing. The sensitivity of testing is nearly 100% for all patients with ADPKD who are age 30 years or older and for younger patients with PKD1 mutations; these criteria are only 67% sensitive for patients with PKD2 mutations who are younger than age 30 years. Large echogenic kidneys without distinct macroscopic cysts in an infant/child at 50% risk for ADPKD are diagnostic. In the absence of a family history of ADPKD, the presence of bilateral renal enlargement and cysts, with or without the presence of hepatic cysts, and the absence of other manifestations suggestive of a different renal cystic disease provide presumptive, but not definite, evidence for the diagnosis.

Molecular genetic testing by linkage analysis or direct mutation screening is available clinically; however, genetic heterogeneity is a significant complication to molecular genetic testing. Sometimes a relatively large number of affected family members need to be tested in order to establish which one of the two possible genes is responsible within each family. The large size and complexity of PKD1 and PKD2 genes, as well as marked allelic heterogeneity, present obstacles to molecular testing by direct DNA analysis. In the research setting, mutation detection rates of 50-75% have been obtained for PKD1 and ~75% for PKD2. Clinical testing of the PKD1 and PKD2 genes by direct sequence analysis is now available, with a detection rate for disease-causing mutations of 50-70%.

Genetic counseling may be helpful for families at risk for polycystic kidney disease.

Treatment

Although a cure for PKD is not available, treatment can ease the symptoms and prolong life.

Pain: Over-the-counter pain medications, such as paracetamol (acetaminophen) can relieve pain. For most but not all cases of severe pain, surgery to shrink cysts can relieve pain in the back and flanks. However, surgery provides only temporary relief and usually does not slow the disease's progression toward kidney failure.

Urinary tract infections: Patients with PKD tend to have frequent urinary tract infections, which can be treated with antibiotics. Early treatment is important, because infection can spread from the urinary tract to the cysts in the kidneys. Cyst infections are difficult to treat because many antibiotics do not penetrate into the cysts. However, some antibiotics are effective.

High blood pressure: Keeping blood pressure under control can slow the effects of PKD. Lifestyle changes including low salt diet and various medications especially ACE inhibitors and angiotensin receptor blockers can lower high blood pressure. Recommended target BP in these patients is 130/80 mm Hg or lower.

Chronic Kidney Disease Progression: A very exciting new development in ADPKD treatment involves the use of Tolvaptan, a selective vasopressin antagonist, and research shows that this compound may limit cyst growth, reduce symptoms and most importantly, delay the progression of chronic kidney disease.[10]

End-stage renal disease: There are two options for replacing kidney functions: dialysis or transplantation. Healthy (non-PKD) kidneys transplanted into PKD patients do not develop cysts.[11]

Prognosis

Despite significant research, prognosis of this disease has changed little over time. It is suggested that avoidance of caffeine may prevent cyst formation. Although not well-proven, treatment of hypertension and a low protein diet may slow progression of the disease.

Between PKD1 and PKD2, the former has the worse prognosis.

References

^ a b c Grantham, Jared (Oct 2 2008). "Autosomal Dominant Polycystic Kidney Disease". New England Journal of Medicine 359: 1477–1485. doi:10.1056/NEJMcp0804458. http://www.nejm.org/doi/full/10.1056/NEJMcp0804458.

^ Torres, Vicente (20 May 2009). "Autosomal dominant polycystic kidney disease: the last 3 years". Kidney International 76 (2): 149–168. doi:10.1038/ki.2009.128. http://www.nature.com/ki/journal/v76/n2/full/ki2009128a.html.

^ DALGAARD OZ (1957). "Bilateral polycystic disease of the kidneys; a follow-up of two hundred and eighty-four patients and their families". Acta Med. Scand. Suppl. 328: 1–255. PMID 13469269.

^ Zerres K, Mücher G, Becker J, et al. (1998). "Prenatal diagnosis of autosomal recessive polycystic kidney disease (ARPKD): molecular genetics, clinical experience, and fetal morphology". Am. J. Med. Genet. 76 (2): 137–44. doi:10.1002/(SICI)1096-8628(19980305)76:2<137::AID-AJMG6>3.0.CO;2-Q. PMID 9511976.

^ Calvet, James P. (Oct 2002). "Cilia in PKD—Letting It All Hang Out". Journal of the American Society of Nephrology 13 (10): 2614–16. PMID 12239253. http://jasn.asnjournals.org/cgi/content/full/13/10/2614.

^ Yoder BK, Hou X, Guay-Woodford LM (Oct 2002). "The Polycystic Kidney Disease Proteins, Polycystin-1, Polycystin-2, Polaris, and Cystin, Are Co-Localized in Renal Cilia". Journal of the American Society of Nephrology 13 (10): 2508–2516. doi:10.1097/01.ASN.0000029587.47950.25. PMID 12239239. http://jasn.asnjournals.org/cgi/content/full/13/10/2508?ijkey=e0e1998129b978c2097e67e9eb70fc0f87fb5810.

^ First Aid for the USMLE. 2011, pg. 472

^ Reeders ST (1992). "Multilocus polycystic disease". Nat. Genet. 1 (4): 235–7. doi:10.1038/ng0792-235. PMID 1338768.

^ Badano, Jose L.; Norimasa Mitsuma, Phil L. Beales, Nicholas Katsanis (September 2006). "The Ciliopathies : An Emerging Class of Human Genetic Disorders". Annual Review of Genomics and Human Genetics 7: 125–148. doi:10.1146/annurev.genom.7.080505.115610. PMID 16722803. http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.genom.7.080505.115610. Retrieved 2008-06-15.

^ UKidney - Internet School of Nephrology: Tolvaptan

^ http://transplant.surgery.ucsf.edu/conditions--procedures/polycystic-kidney-disease.aspx

Additional images

Adult polycystic kidney.

출처: http://en.wikipedia.org/wiki/Autosomal_dominant_polycystic_kidney

Figure 3.5 Adult onset polycyctic kidney disease and microsatellite polymorphism

ㆍminisatellite도 역시 70 % 정도에서 heteozygosity를 보인다(Fig. 3.6).

Figure 3.6 Minisatellite and paternity test

Figure 3.7 DNA fingerprinting

ㆍ여러 개의 minisatellite 다형성을 동시에 관찰하는 소위 DNA fingerprint 법이 개발되어 법의학적으로 유용하게 사용되고 있다(Fig. 3.7).

1.1 DNA 다형성을 보는 기술

ㆍDNA 다형성을 확인하기 위해 두 가지 방법을 주로 사용한다. 그 하나는 southern analysis 이고 다른 하나는 PCR 이다.

※southern analysis와 PCR의 원리를 알아봅시다(Fig. 3.8, Fig. 3.9)

Figure 3.8 Southern analysis

Figure 3.9 PCR

ㆍPCR의 장점은 적은 양의 DNA 시료가 필요하다는 점이며, 증폭한 DNA를 제한효소로 다시 소화하여 자리 다형성을 확인할 수 있다.

2. 직접적 돌연변이 유전자 분석

ㆍ간접적 방법은 가족 중 여러 사람의 DNA 시료를 필요로 할뿐더러, 질병 유전자의 추적에 사용할 정확한 다형성 marker의 선택이 요구되어 진다.

ㆍ또한 marker와 질병 유전자 사이의 거리에 따라서 에러가 발생할 수 있다.

ㆍ반면에 직접적인 방법은 적은 수의 시료가 필요하며, 돌연변이를 확인함으로서 질병의 진단을 확실히 내릴 수 있다.

Figure 3.11 PCR of beta-globin gene and digestion

ㆍ길이 돌연변이는 southern analysis 혹은 PCR로 확인될 수 있다(Fig. 3.10).

Figure 3.10 Simultaneous PCR of nine segments of DMD gene

ㆍ점 돌연변이는 RFLP, allele specific oligonucleotide probe, DNA sequencing 등에 의해 확인할 수 있다(Fig. 3.11).

ㆍAllele specific oligonucleotide(ASO) probe는 17-30 nucleotide로 된 합성 DNA로서 정상적인 DNA 혹은 비정상적 DNA에 상보적인 염기배열을 갖고 있다. hybridization이 되는지 안 되는지를 보아서 정상, 비정상을 진단하게 된다. homozygous 인지 heterozygous 인지를 구분할 수 있다(Fig. 3.12).

ㆍDNA sequencing : 해당 유전자의 염기배열을 조사하는 것(Fig. 3.13)

Figure 3.12 Dot blot analysis using radiolabelled allele-specific oligonucleotide probes

※DNA sequencing 방법 중 chain termination 방법의 원리와 실제 적용에 대해 토의해 봅시다.

Figure 3.13 Chain termination method

Figure 3.14 DNA sequencing in a haemophilia A patient and normal control