Name: genetic analysis of hereditary transthyretin ala97ser related amyloidosis
Uploaded: 2018-06-09T15:00:00.000+00:00
Duration: 6 min 33 s
Description: Watch this Scientific Journal Video about Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis at JoVE.com

우리가 실험에서 그녀의 도움에 대 한 미스 신 재미 우 감사 하고자 합니다. 이 연구는 장 공 의학 연구 프로그램 (CMRPG3C0371, CMRPG3C0372, CMRPG3C0373)와 IRB 100-4470A3, 104-2462A3, 대만에서 교부 금에 의해 지원 되었다.

임상 실험실 개선 개정 (CLIA) 1988 년의 기관 검토 위원회의 장 궁 메모 리얼 병원에서 승인 하는 규정 요구 사항에 따라 실시 되었다 테스트 실험실에서 수행 하 고 대학 (라이센스 번호 100-4470A3 및 104-2462A3). 동의 모든 환자에서 얻은 했다.
1. 혈액 표본 수집
<ol><li>상업적으로 사용할 수 있는 치료를 EDTA 튜브로 전체 혈액을 수집 합니다. 부드럽게 혼합 하 고 처리까지 4 ° C에서 혈액 샘플을 저장 합니다.</li>
</ol>2. 말 초 혈액에서 DNA 추출
DNA 추출 키트를 사용 하 여 게놈 DNA 추출 ( 재료의 표참조).
<ol><li>4.0 mL 혈액 샘플을 솔루션 1의 10 mL를 추가 합니다. 10 분 동안 얼음에 샘플을 품 어.</li>
	<li>4 ° c.에서 5 분 동안 2000 x g에서 샘플을 원심 제거 하 고 상쾌한을 신중 하 게 삭제. 솔루션 1의 3 mL에 펠 릿을 resuspend.</li>
	<li>다시 4 ° C에서 5 분 동안 2000 x g에서 샘플을 원심 하 고 상쾌한 삭제. 솔루션 2의 3 mL에 펠 릿을 resuspend.</li>
	<li>100 µ g/mL의 최종 농도 고 잘 섞어 나 재고 솔루션 (225 mg/mL)의 10 µ L를 추가 합니다. 37 ° C에서 밤새 품 어.</li>
	<li>10 분 추가 0.8 mL 솔루션 3의 샘플에 대 한 얼음에 튜브를 진정 하 고 반전 3-5 번. 5 분 동안 얼음에 샘플을 놓습니다.</li>
	<li>4 ° c.에 15 분 동안 3000 x g에서 샘플을 원심 조심 스럽게 15 mL 원뿔 메 마른 분리기 관에는 상쾌한 전송.</li>
	<li>20 µ g/mL의 최종 농도를 RNase 재고 솔루션 (10 mg/mL)의 6 µ L를 추가 합니다. 15 분 동안 37 ° C에서 품 어.</li>
	<li>부드럽게 여러 번 DNA를 침전을 반전 하 여 이소프로필 알코올과 혼합의 2.5 mL를 철저 하 게 추가 하는 방법 (흰색, 부드러운 재질의 가닥 형성). 큰 구멍 피펫으로 사용 하 여 새로운 1.5 mL 원심 분리기 튜브 DNA 비례적 전송.</li>
	<li>4 ° c.에 3 분 동안 12000 x g에서 원심 분리기 상쾌한 신중 하 게 삭제 합니다. 씻어 DNA 펠 릿의 70% 에탄올 500 µ L을 추가 하 여.</li>
	<li>1 분 동안 12000 x g에서 centrifuge 고 상쾌한 삭제. 공기 건조 실 온에서 DNA 펠 릿. DdH2O의 100 µ L을 추가 하 고 65 ° C 15 분 DNA resuspend에서 품 어.</li>
</ol>3. 게놈 DNA 정량화
분 광 광도 계를 사용 하 여 ( 재료의 표참조) 게놈 DNA의 품질과 수량을 감지 하.
<ol><li>분 광 광도 계 컴퓨터에 연결 된 컴퓨터에 로그인 합니다. 소프트웨어를 엽니다.</li>
	<li>분 광 광도 계 기계 초기화:<ol>
<li>분 광 광도 계 소프트웨어에 "핵 산"을 클릭 합니다.</li>
		<li>일회용 종이 지우기와 순화 된 물 받침대를 청소.</li>
	</ol>
</li>
	<li>분 광 광도 계 빈:<ol>
<li>주 춧 대에 순화 된 물의 2 µ L을 로드 합니다.</li>
		<li>분 광 광도 계의 윗부분을 낮출 하 고 "빈" 보정을 클릭 합니다.</li>
		<li>완료 되 면 위 팔을 들어올린 닦기와 받침대를 건조.</li>
	</ol>
</li>
	<li>측정 샘플:<ol>
<li>샘플 종류에 "DNA 50"을 클릭 합니다.</li>
		<li>주 춧 대에 DNA 샘플의 2 µ L을 로드 합니다.</li>
		<li>낮은 팔 위쪽을 A260/A280 비율과 샘플의 농도 측정 하기 위해 "측정"을 클릭 합니다.</li>
		<li>지우기와 순화 된 물으로 각 실행 사이 받침대를 청소.</li>
	</ol>
</li>
	<li>"Print Screen" 데이터 수집을 클릭 합니다.</li>
</ol>4. 유전자 돌연변이 분석
PCR4대상 DNA를 증폭. 25 µ L 반응에서 PCR을 수행 DNA 중 합 효소를 사용 하 여 혼합 키트 ( 재료의 표참조). 표 1 에서 intronic 뇌관 TTR 유전자를 보여준다.
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>유전자</td>
			<td>뇌관 순서</td>
		</tr>
<tr>
<td>Exon1</td>
			<td>F: 5'-TCAGATTGGCAGGGATAAGC-3' R: 5'-GCAAAGCTGGAAGGAGTCAC-3'</td>
		</tr>
<tr>
<td>Exon2</td>
			<td>F: 5'-TCTTGTTTCGCTCCAGATTTC-3' R: 5'-TCTACCAAGTGAGGGGCAAA-3'</td>
		</tr>
<tr>
<td>Exon3</td>
			<td>F: 5'-GTGTTAGTTGGTGGGGGTGT-3' R: 5'-TGAGTAAAACTGTGCATTTCCTG-3'</td>
		</tr>
<tr>
<td>Exon4</td>
			<td>F: 5'-GACTTCCGGTGGTCAGTCAT-3' R: 5'-GCGTTCTGCCCAGATACTTT-3'</td>
		</tr>
</tbody></table>표 1입니다. TTR 유전자 intronic 뇌관. (NCBI 참고 순서: NC_000018.10)
<ol><li>0.2 mL PCR 튜브를 표 2 에 시 약을 추가 합니다.</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>구성 요소</td>
			<td>볼륨 (µ L)</td>
			<td>최종 농도</td>
		</tr>
<tr>
<td>10 x 버퍼</td>
			<td>2.5</td>
			<td>1 x</td>
		</tr>
<tr>
<td>MgCl2 (25 mM)</td>
			<td>1.5</td>
			<td>1.5 m m</td>
		</tr>
<tr>
<td>360 GC 증강</td>
			<td>1</td>
			<td>-</td>
		</tr>
<tr>
<td>360 DNA 중 합 효소</td>
			<td>0.2</td>
			<td>2 U/반응</td>
		</tr>
<tr>
<td>dNTP 믹스 (각 25 mM)</td>
			<td>2</td>
			<td>2 m m</td>
		</tr>
<tr>
<td>뇌관 F (10 µ M)</td>
			<td>1</td>
			<td>0.4 Μ M</td>
		</tr>
<tr>
<td>뇌관 R (10 µ M)</td>
			<td>1</td>
			<td>0.4 Μ M</td>
		</tr>
<tr>
<td>DNA (100 ng)</td>
			<td>2</td>
			<td></td>
		</tr>
<tr>
<td>PCR-등급 물</td>
			<td>13.8</td>
			<td>-</td>
		</tr>
<tr>
<td>총 볼륨</td>
			<td>25</td>
			<td>-</td>
		</tr>
</tbody></table>표 2입니다. PCR 반응 조건입니다.
<ol start="2"><li>부드럽게 혼합 하 고 짧게 튜브의 하단에 모든 구성 요소를 수집 하는 벤치탑 원심 분리기에 원심. 튜브는 thermocycler 장소.</li>
	<li>30 사이클에 대 한 PCR을 수행 합니다. 각 주기 30 변성 단계 이루어져 94 ° C, 30에 대 한 단련 하는 뇌관에서 s 58 ° C, 및 45에 대 한 중 합 효소 확장에서 s s 72 ° C에서 ( 표 3참조).</li>
</ol><table fo:keep-together.within-page="1"><tbody>
<tr>
<td>단계</td>
			<td>온도 (° C)</td>
			<td>시간</td>
			<td>사이클</td>
		</tr>
<tr>
<td>초기 변성</td>
			<td>95</td>
			<td>5 분</td>
			<td>1</td>
		</tr>
<tr>
<td>DNA 변성 단계</td>
			<td>94</td>
			<td>30 s</td>
			<td>30 사이클</td>
		</tr>
<tr>
<td>뇌관 어 닐 링의 단계</td>
			<td>58</td>
			<td>30 s</td>
			<td>30 사이클</td>
		</tr>
<tr>
<td>중 합 효소 확장 단계</td>
			<td>72</td>
			<td>45 s</td>
			<td>30 사이클</td>
		</tr>
<tr>
<td>포스트 신장 단계</td>
			<td>72</td>
			<td>10 분</td>
			<td>1</td>
		</tr>
<tr>
<td>PCR의 끝 자전거</td>
			<td>4</td>
			<td>무기한</td>
			<td></td>
		</tr>
</tbody></table>표 3입니다. 사이클링 증폭 PCR 제품을 위한 조건.
<ol start="4"><li>젤 전기 이동 법으로 시각화 하 여 PCR 제품을 확인:<ol>
<li>1.2 %agarose 젤 준비:<ol>
<li>0.5의 100 mL의 agarose 분말 1.2 g 혼합 microwavable 플라스 크에 x TBE. 전자 레인지는 agarose는 완전히 용 해 될 때까지 2 분. 55 ° c agarose 혼합물을 진정</li>
			<li>Ethidium 평범한 사람 해결책 (10mg/mL)의 2 µ L agarose 혼합물에 추가 하 고 부드럽게 혼합. 약 5-7 m m 젤 트레이에 agarose 혼합물을 부 어 다음 장소에는 comb(s)를 추가 합니다. (적어도 30 분)을 공고히 하 고 comb(s)를 조심 스럽게 제거를 하실 수 있습니다.</li>
		</ol>
</li>
		<li>샘플을 로드 하 고 실행 하는 agarose 젤:<ol>
<li>트레이 젤 전기 이동 법 셀에 놓습니다. 0.5 전기 셀 채우기 x TBE 젤 커버까지.</li>
			<li>신중 하 게 로드 2 µ L 100 bp DNA의 분자 크기 표식으로 젤의 한 차선에 ( 재료의 표참조) 사다리. 부하 염료/샘플 혼합 (5 µ L PCR 제품 믹스의 로드 염료 x 1 µ L 6와) 추가 차선에.</li>
			<li>전원 공급 장치를 켭니다. 100 V에서 25 분 젤을 실행 합니다.</li>
			<li>젤 전기 이동 법 셀에서 제거 합니다. 자외선 이미징 시스템에서에서 DNA 파편을 시각화.</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. DNA 시퀀싱
<ol><li>상업 키트를 사용 하 여 PCR 제품을 정화 하 고 상업적으로 또는 자동 시퀀서는 시퀀싱 ( 재료의 표참조).</li>
	<li>뉴클레오티드 시퀀스 뉴클레오티드 데이터베이스10뉴클레오티드 쿼리를 비교 하는 NCBI 폭발 서버에 제출 합니다. 결과 표시 후 시퀀스 정렬을 수행 하 고 예상된 돌연변이 식별 합니다.</li>
</ol>

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A., Dispenzieri, A., Sher, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+and+treatment+of+cardiac+amyloidosis.">Pathophysiology and treatment of cardiac amyloidosis.</a> Nature Reviews Cardiology. 12 (2), 91-102 (2015).</li><li>Vrana, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+diagnosis+and+typing+of+systemic+amyloidosis+in+subcutaneous+fat+aspirates+by+mass+spectrometry-based+proteomics.">Clinical diagnosis and typing of systemic amyloidosis in subcutaneous fat aspirates by mass spectrometry-based proteomics.</a> Haematologica. 99 (7), 1239-1247 (2014).</li><li>Nakamura, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+new+transthyretin+variant+(Ile49)+in+familial+amyloidotic+polyneuropathy+using+electrospray+ionization+mass+spectrometry+and+nonisotopic+RNase+cleavage+assay.">Identification of a new transthyretin variant (Ile49) in familial amyloidotic polyneuropathy using electrospray ionization mass spectrometry and nonisotopic RNase cleavage assay.</a> Human Heredity. 49 (4), 186-189 (1999).</li><li>Ueda, M., Ando, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+transthyretin+amyloidosis+therapy.">Recent advances in transthyretin amyloidosis therapy.</a> Translational Neurodegeneration. 3, 19 (2014).</li><li>Brown, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+testing+improves+identification+of+transthyretin+amyloid+(ATTR)+subtype+in+cardiac+amyloidosis.">Genetic testing improves identification of transthyretin amyloid (ATTR) subtype in cardiac amyloidosis.</a> Amyloid. 24 (2), 92-95 (2017).</li><li>Tasaki, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+wild-type+and+mutated+transthyretins.">Rapid detection of wild-type and mutated transthyretins.</a> Annals of Clinical Biochemistry. 53 (4), 508-510 (2015).</li><li>Plante-Bordeneuve, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+pitfalls+in+sporadic+transthyretin+familial+amyloid+polyneuropathy+(TTR-FAP).">Diagnostic pitfalls in sporadic transthyretin familial amyloid polyneuropathy (TTR-FAP).</a> Neurology. 69 (7), 693-698 (2007).</li><li>Hsu, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective,+observational+study+of+patients+with+uncommon+distal+symmetric+painful+small-fiber+neuropathy.">A prospective, observational study of patients with uncommon distal symmetric painful small-fiber neuropathy.</a> PLoS ONE. 12 (9), 0183948 (2017).</li><li>Salvi, F., Pastorelli, F., Plasmati, R., Bartolomei, I., Dall'Osso, D., Rapezzi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+and+phenotypic+correlation+in+an+Italian+population+of+hereditary+amyloidosis+TTR-related+(HA-TTR):+Clinical+and+neurophysiological+aids+to+diagnosis+and+some+reflections+on+misdiagnosis.">Genotypic and phenotypic correlation in an Italian population of hereditary amyloidosis TTR-related (HA-TTR): Clinical and neurophysiological aids to diagnosis and some reflections on misdiagnosis.</a> Amyloid. 19, 58-60 (2012).</li><li>Suhr, O. B., Lundgren, E., Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+mutation,+two+distinct+disease+variants:+Unravelling+the+impact+of+transthyretin+amyloid+fibril+composition.">One mutation, two distinct disease variants: Unravelling the impact of transthyretin amyloid fibril composition.</a> Journal of Internal Medicine. 281 (4), 337-347 (2017).</li><li>Vil&#224;-Rico, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+post-translational+modifications+in+human+serum+transthyretin+associated+with+familial+amyloidotic+polyneuropathy+by+targeted+LC-MS+and+intact+protein+MS.">Quantitative analysis of post-translational modifications in human serum transthyretin associated with familial amyloidotic polyneuropathy by targeted LC-MS and intact protein MS.</a> Journal of Proteomics. 127, 234-246 (2015).</li></ol>

저자는 공개 없다.

프로토콜 내에서 두 가지 중요 한 단계가 있습니다. 첫째, 백혈구 세포의 충분 한 수를 위해, hemodiluted 견본 피할된11이어야 한다. 둘째, 적절 한 PCR 뇌관의 사용이 이다 신뢰할 수 있는 결과12를 기본. 디자인 뇌관4,13; 뇌관 폭발 웹 도구를 사용 하는 우리 4 개의 TTR exons의 각 측에 40의 기본적인 쌍의 최소 처리 되어야 합...

Transthyretin (TTR) amyloidosis (ATTR) 조직의 유전 amyloidosis1, 가장 일반적인 형태 이며 transthyretin (TTR) 유전자2상 염색체 지배적 상속 된 돌연변이 의해 발생할 수 있습니다. TTR 돌연변이 tetrameric 단백질 구조를 불안정 하 게 하 고 녹말 체 소2로 재어셈블합니다 단위체로 그것의 분리 이어질. 100 amyloidogenic TTR 변이 보고 전세계1되었습니다. 중 합 효소 연쇄 반응 (PCR) 방법으로 유전자 분석 TTR 점 돌연변이의 존재를 확인 하 고 피하는 남쪽 오 점3에 비해 방사성 레이블이 프로브의 처리를 포함 하는 장점이 있다. PCR은 현대 과학4에 수많은 분야에 적용 된, 쉽게, 저렴 한, 빠르고 신뢰할 수 있는 기술입니다.
이 진보 및 치명적인 질병의 조기 진단의 phenotypic이 주어진 도전 이다. 관심과 ATTR 신흥 치료5의 초기 발현에 광범위 한 인식 증가, TTR 유전자 검사를 포함 한 적절 한 진단 연구는 따라서 비판적으로 예 후를 개선 하기 위해 근본적인. 또한, 다른 변이 특성의 다른 penetrance, 발병 연령, 진행, 질병의 심각도, 메디아 생존, 간 이식, 또는 TTR 안정기2,6, 효능의 패턴에 연관 된 고 가변도 신경학 적인 참여의 유전 상담7,,89에 대 한 큰 의미를가지고. 게다가, 매우 정확한 유전자 검사는 두 가지 유형의 속성을 구분 하는 유일한 도구: 유전 (돌연변이) 및 야생 유형 (비 돌연변이 형태, 치 조직의 amyloidosis, 특수)7. 그것은 치료2넓게 변화 하기 때문에, 속성의 종류를 확인 하는 것이 필수적입니다. 그러므로, TTR 유전자 검사의 stepwise 프로토콜을 설명 하는 증가 필요는 없다.
돌연변이 검출 하는 분자 접근 방식은 예를 들어 Ala97Ser, 대만, 가장 일반적인 발병 변이 사용 하 여 그림 것입니다. 수정 DNA 추출 단계에서 사용 되는 세 가지 솔루션의 양을 줄일 하 고 충분 한 양의 DNA 생성 합니다. 이 프로토콜에서 모든 4 개의 TTR exons 지역 5' 상류 3' 하류, 발기인, introns를 포함 하 여 동안, 분석 되었다 그리고 번역 되지 않은 영역 (UTR) 시퀀싱 하지 했다.

<table><tbody><tr><td>EDTA-treated tubes</td><td>BD</td><td></td><td></td></tr><tr><td>DNA Extraction Kit</td><td>Stratagen</td><td>200600</td><td></td></tr><tr><td>NanoDrop ND2000 spectrophotometer&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>NanoDrop 2000</td><td></td></tr><tr><td>Delicate Task Wipers</td><td>Kimberly-Clark</td><td>Kimtech Science Kimwipes</td><td></td></tr><tr><td>AmpliTaq Gold 360 DNA Polymerase kit</td><td>Applied Biosystems</td><td>4398823</td><td></td></tr><tr><td>TTR gene intronic primers&amp;nbsp;</td><td>Exon1</td><td>F: 5&amp;rsquo;-TCAGATTGGCAGGGATAAGC-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon1</td><td>R: 5&amp;rsquo;-GCAAAGCTGGAAGGAGTCAC-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon2</td><td>F: 5&amp;rsquo;-TCTTGTTTCGCTCCAGATTTC-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon2</td><td>R: 5&amp;rsquo;-TCTACCAAGTGAGGGGCAAA-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon3</td><td>F: 5&amp;rsquo;-GTGTTAGTTGGTGGGGGTGT-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon3</td><td>R: 5&amp;rsquo;-TGAGTAAAACTGTGCATTTCCTG-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon4</td><td>F: 5&amp;rsquo;-GACTTCCGGTGGTCAGTCAT-3&amp;rsquo;</td><td></td></tr><tr><td></td><td>Exon4</td><td>R: 5&amp;rsquo;-GCGTTCTGCCCAGATACTTT-3&amp;rsquo;</td><td></td></tr><tr><td>thermocycler</td><td>Applied Biosystems</td><td>GeneAmp PCR System 9700</td><td></td></tr><tr><td>electrophoresis cell&amp;nbsp;</td><td>ADVANCE</td><td>Mupid-2plus</td><td></td></tr><tr><td>DNA ladder</td><td>Protech</td><td>PT-M1-100</td><td></td></tr><tr><td>dye</td><td>BioLabs</td><td>B7021</td><td></td></tr><tr><td>AlphaImager EC</td><td>Alpha Innotech</td><td>AlphaImager EC</td><td></td></tr><tr><td>automatic sequencer&amp;nbsp;</td><td>Applied Biosystems</td><td>3730xl DNA Analyzer</td><td></td></tr></tbody></table>

genetic analysis of hereditary transthyretin ala97ser related amyloidosis

유전자 검사 이며 유전 transthyretin에 대 한 가장 신뢰할 수 있는 테스트 관련 amyloidosis transthyretin amyloidosis (ATTR)의 대부분의 경우에서 수행 되어야 합니다. 속성은 유형이 다른 고기;와 희귀 하지만 치명적인 질병 따라서, 진단 때로는 지연 됩니다. 관심과 ATTR 신흥 치료의 초기 발현에 광범위 한 인식 증가 함께 transthyretin (TTR) 유전자를 포함 하 여 적절 한 진단 연구, 테스트, 종류를 확인 하 고 ATTR의 변종 따라서 예 후를 개선 하기 위해 근본적인. 중 합 효소 연쇄 반응 (PCR) 방법으로 유전자 분석 TTR 점 돌연변이의 존재를 훨씬 더 신속 하 게 확인 하 고 남쪽 오 점 등 기존 방법 보다 안전. 여기, 우리가 ATTR Ala97Ser 돌연변이, 대만에서 가장 일반적인 발병 돌연변이의 유전자 확인을 보여 줍니다. 프로토콜 구성 4 가지 주요 단계: 수집 전 혈 견본, DNA 추출, PCR와 모든 4 개의 TTR exons의 유전자 분석 및 DNA 연속.

Agarose 젤 전기 이동 법 두 환자와 exon TTR 유전자 (그림 1)의 4에 대 한 454 bp의 PCR 제품을 포함 한 예상된 크기에의 한 건강 한 개별 계시 밴드.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57743/57743fig1.jpg"> 그림 1 . 젤 전기 이동 법 묘사 PCR 증폭 TTR 유전자. 일반: 건강 한 ?...

Watch this Scientific Journal Video about Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis at JoVE.com

Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

여기, 우리는 예를 들어 Ala97Ser, 대만, 가장 일반적인 발병 변이 사용 하 여 유전 transthyretin amyloidosis의 진단에 대 한 점 돌연변이의 존재를 확인 하는 프로토콜 제시.

유전 Transthyretin Ala97Ser의 유전 분석 관련 Amyloidosis

Genetic testing is the most reliable test for hereditary transthyretin related amyloidosis and should be performed in most cases of transthyretin ...

genetic-analysis-hereditary-transthyretin-ala97ser-related

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

7.7K 조회수.  Chang Gung Memorial Hospital Linkou Medical Center and Chang Gung University College of Medicine. 여기, 우리는 예를 들어 Ala97Ser, 대만, 가장 일반적인 발병 변이 사용 하 여 유전 transthyretin amyloidosis의 진단에 대 한 점 돌연변이의 존재를 확인 하는 프로토콜 제시.

비디오: Genetic Analysis of Hereditary Transthyretin Ala97Ser Related Amyloidosis

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (A&#946;) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

Here, we describe an innovative method to determine complement receptor 1 (CR1) length polymorphisms for use in several applications, particularly the assessment of susceptibility to diseases such as Alzheimer&#39;s disease (AD). This method could be useful to better understand the role of CR1 isoforms in the pathogenesis of AD.

보체 수용체 1 길이 다형성 유전자 타이핑을위한 고해상도 Melting PCR : 알츠하이머 병 유전자 감수성 평가를위한 혁신적인 도구

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

연구

유전학

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

다음-세대 시퀀싱과 생물 정보학 파이프라인 헌법 질병의 유전 결정 요인 평가 대상

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

Thromboxane A2 수용 체 유전자의 C924T 다형성을 공부 하는 방법

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Confocal 현미경 검사 법 그리고 형광 일생 화상 진 찰 Hyperspectral에 의해 발광 활용된 Oligothiophenes로 물 녹말 체 조직 이미징

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

생화학

Determining the Likelihood of Variant Pathogenicity Using Amino Acid-level Signal-to-Noise Analysis of Genetic Variation

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. While this has led to increased identification of likely pathogenic mutations associated with genetic syndromes, it has also dramatically increased the number of incidentally found genetic variants of unknown significance (VUS). Determining the clinical significance of these variants is a major challenge for both scientists and clinicians. An approach to assist in determining the likelihood of pathogenicity is signal-to-noise analysis at the protein sequence level. This protocol describes a method for amino acid-level signal-to-noise analysis that leverages variant frequency at each amino acid position of the protein with known protein topology to identify areas of the primary sequence with elevated likelihood of pathologic variation (relative to population &#34;background&#34; variation). This method can identify amino acid residue location &#34;hotspots&#34; of high pathologic signal, which can be used to refine the diagnostic weight of VUSs such as those identified by next generation genetic testing.

Amino acid-level signal-to-noise analysis determines the prevalence of genetic variation at a given amino acid position normalized to background genetic variation of a given population. This allows for identification of variant &#34;hotspots&#34; within a protein sequence (signal) that rises above the frequency of rare variants found in a population (noise).

유전 변이의 아미노산 수준 신호-잡음 분석을 사용 하 여 Variant Pathogenicity의 가능성 확인

Advancements in the cost and speed of next generation genetic sequencing have generated an explosion of clinical whole exome and whole genome testing. ...

Mapping Alzheimer's Disease Variants to Their Target Genes Using Computational Analysis of Chromatin Configuration

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, because the majority of the genome-wide significant (GWS) loci fall onto the non-coding genome, the functional impact of many remain unknown. Three-dimensional chromatin interactions identified by Hi-C or its derivatives can provide useful tools to annotate these loci by linking non-coding variants to their actionable genes. Here, we outline a protocol to map GWAS non-coding variants to their putative genes using Alzheimer&#39;s disease (AD) GWAS and Hi-C datasets from human adult brain tissue. Putative causal single-nucleotide polymorphisms (SNPs) are identified by application of fine-mapping algorithms. SNPs are then mapped to their putative target genes using enhancer-promoter interactions based on Hi-C. The resulting gene set represents AD risk genes, as they are potentially regulated by AD risk variants. To garner further biological insights into molecular mechanisms underlying AD, we characterize AD risk genes using developmental brain expression data and brain single-cell expression profiles. This protocol can be expanded to any GWAS and Hi-C datasets to identify putative target genes and molecular mechanisms underlying various human traits and diseases.

We present a protocol to identify functional implications of non-coding variants identified by genome-wide association studies (GWAS) using three-dimensional chromatin interactions.

크로마틴 구성의 전산 분석을 사용하여 알츠하이머 병 변이체를 대상 유전자에 매핑

Genome-wide association studies (GWAS) have successfully identified hundreds of genomic loci that are associated with human traits and disease. However, ...

Novel Atomic Force Microscopy Based Biopanning for Isolation of Morphology Specific Reagents against TDP-43 Variants in Amyotrophic Lateral Sclerosis

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson&#8217;s disease and beta-amyloid and tau in Alzheimer&#8217;s disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.

Using atomic force microscopy in combination with biopanning technology we created a negative and positive biopanning system to acquire antibodies against disease-specific protein variants present in any biological material, even at low concentrations. We were successful in obtaining antibodies to TDP-43 protein variants involved in Amyotrophic Lateral Sclerosis.

루게릭 병에 TDP-43 변종 형태론 특정 시약의 분리에 대한 소설 원자 힘 현미경 기반 Biopanning

Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in ...

생체공학

Investigating the Pathogenesis of MYH7 Mutation Gly823Glu in Familial Hypertrophic Cardiomyopathy using a Mouse Model

Familial hypertrophic cardiomyopathy (HCM, OMIM: 613690) is the most common cardiomyopathy in China. However, the underlying genetic etiology of HCM remains elusive.
We previously identified a myosin heavy chain 7 (MYH7) gene heterozygous variant, NM_000257.4: c.G2468A (p.G823E), in a large Chinese Han family with HCM. In this family, variant G823E cosegregates with an autosomal dominant disorder. This variant is located in the lever arm domain of the neck region of the MYH7 protein and is highly conserved among homologous myosins and species. To verify the pathogenicity of the G823E variant, we produced a C57BL/6N mouse model with a point mutation (G823E) at the mouse MYH7 locus with CRISPR/Cas9-mediated genome engineering. We designed gRNA targeting vectors and donor oligonucleotides (with targeting sequences flanked by 134 bp of homology). The p.G823E (GGG to GAG) site in the donor oligonucleotide was introduced into exon 23 of MYH7 by homology-directed repair. A silenced p.R819 (AGG to CGA) was also inserted to prevent gRNA binding and re-cleavage of the sequence after homology-directed repair. Echocardiography revealed left ventricular posterior wall (LVPW) hypertrophy with systole in MYH7 G823E/- mice at 2 months of age. These results were likewise validated by histological analysis (Figure 3).
These results demonstrate that the G823E variant plays an important role in the pathogenesis of HCM. Our findings enrich the spectrum of MYH7 variants linked to familial HCM and may provide guidance for genetic counseling and prenatal diagnosis in this Chinese family.

Based on the familial hereditary cardiomyopathy family found in our clinical work, we created a C57BL/6N mouse model with a point mutation (G823E) at the mouse MYH7 locus through CRISPR/Cas9-mediated genome engineering to verify this mutation.

마우스 모델을 사용한 가족성 비후성 심근병증에서 MYH7 돌연변이 Gly823Glu의 발병기전 조사

Familial hypertrophic cardiomyopathy (HCM, OMIM: 613690) is the most common cardiomyopathy in China. However, the underlying genetic etiology of HCM ...

Demonstration of the Sequence Alignment to Predict Across Species Susceptibility Tool for Rapid Assessment of Protein Conservation

The US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool is a fast, freely available, online screening application that allows researchers and regulators to extrapolate toxicity information across species. For biological targets in model systems such as human cells, mice, rats, and zebrafish, toxicity data are available for a variety of chemicals. Through the evaluation of protein target conservation, this tool can be used to extrapolate data generated from such model systems to thousands of other species lacking toxicity data, yielding predictions of relative intrinsic chemical susceptibility. The latest releases of the tool (versions 2.0-6.1) have incorporated new features that allow for the rapid synthesis, interpretation, and use of the data for publication plus presentation-quality graphics.
Among these features are customizable data visualizations and a comprehensive summary report designed to summarize SeqAPASS data for ease of interpretation. This paper describes the protocol to guide users through submitting jobs, navigating the various levels of protein sequence comparisons, and interpreting and displaying the resulting data. New features of SeqAPASS v2.0-6.0 are highlighted. Furthermore, two use-cases focused on transthyretin and opioid receptor protein conservation using this tool are described. Finally, SeqAPASS&#39; strengths and limitations are discussed to define the domain of applicability for the tool and highlight different applications for cross-species extrapolation.

Here, we present a protocol to utilize the latest version of the US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool. This protocol demonstrates the application of the online tool to rapidly analyze protein conservation and provide customizable and easily interpretable predictions of chemical susceptibility across species.

단백질 보존의 신속한 평가를 위한 종 간 감수성 도구를 예측하기 위한 서열 정렬 입증

The US Environmental Protection Agency Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool is a fast, freely available, online ...

A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to lessen the impact of debilitating conditions but remain elusive in many cases. Only by understanding the causes and pathology of diseases associated with aging, can scientists begin to identify potential therapeutic targets and develop strategies for intervention. The most common age-related conditions are neurodegenerative disorders such as Parkinson's disease and blindness. Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Genome wide association studies have previously identified loci that are associated with increased susceptibility to this disease and identified two regions of interest: complement factor H (CFH) and the 10q26 locus, where the age-related maculopathy susceptibility 2 (ARMS2) and high-temperature requirement factor A1 (HtrA1) genes are located. CFH acts as a negative regulator of the alternative pathway (AP) of the complement system while HtrA1 is an extracellular serine protease. ARMS2 is located upstream of HtrA1 in the primate genome, although the gene is absent in mice. To study the effects of these genes, humanized knock-in mouse lines of Cfh and ARMS2, knockouts of Cfh, HtrA1, HtrA2, HtrA3 and HtrA4 as well as a conditional neural deletion of HtrA2 were generated. Of all the genetically engineered mice produced only mice lacking HtrA2, either systemically or in neural tissues, displayed clear phenotypes. In order to examine these mice thoroughly and systematically, an initial phenotyping schedule was established, consisting of a series of tests related to two main diseases of interest: AMD and Parkinson's. Genetically modified mice can be subjected to appropriate experiments to identify phenotypes that may be related to the associated diseases in humans. A phenotyping regimen with a mitochondrial focus is presented here alongside representative results from the tests of interest.

A reverse-genetics approach to understanding gene families associated with human disease is presented, using mouse as a model system, and the subsequent mouse phenotyping schedule is described. Because mice defective in a gene of interest, HtrA2, manifested Parkinsonian symptoms, the phenotyping regimen is focused on identifying neurological defects.

노화의 인간의 질병에 관여 유전자를 연구에 사용 된 유전자 변형 마우스에 대한 표현형 요법

Age-related diseases are becoming increasingly prevalent and the burden continues to grow as our population ages. Effective treatments are necessary to ...