「全基因組分析」有什麼用?

每個人的身體狀況會因為居住環境、生活習慣而有相當大的差異。但對於同一家族裡的成員來說,相似的基因存在親戚手足之間,彼此未來健康與否,可以從家族病史中看出一些端倪。不過,或許在不久的將來,就可以透過個人的基因資訊,直接評估患病風險,還能篩選適合自己的用藥。

中研院生物醫學科學研究所(簡稱生醫所)所長郭沛恩與研究團隊,正努力推行兩個基因分析計畫:一個針對罕見遺傳疾病;另一個則力求讓大眾問診、用藥更加精確。攝影│張語辰

請問家族病史?

身體不舒服去看醫生時,會填一張病歷表,其中一項常見的問題是:

家族有哪些病史。

疾病的成因,有些是因為先天基因變異,有些是後天環境、飲食、傳染病造成。詢問家族病史的目的在於,相似的基因自祖父母、雙親,再傳到子代,想知道罕見遺傳疾病、甚或癌症、心血管疾病、糖尿病等發生的機會,過往是透過詢問「家族病史」來瞭解。

但未來將有更精確的方式預估患病風險——直接查看基因資訊

圖/wikimedia

罕見遺傳疾病:分析單一基因變異

針對單一基因異常造成的罕見遺傳疾病,郭沛恩團隊使用「全基因組遺傳分析」(full genome analysis) 來分析病因。

這類罕見遺傳疾病,包含先天性異常 (congenital anomalies) 、神經退化性疾​​病 (neurodegenerative disease) 等等。全基因組遺傳分析分成兩個部分,只要從血液、口水取得可供分析的 DNA,就可以進行全基因體定序 (whole genome sequencing),了解該 DNA 片段的鹼基如何排列;以及繪製全基因體圖譜 (whole genome mapping),了解該 DNA 片段在染色體上的位置。

郭沛恩研究團隊的成員,從血液細胞樣本裡抽取 DNA 。攝影│張語辰

首先,「全基因體定序」,是將已發病患者的 23 對染色體中,每一個基因全部都定序。定序完成後,參考已知的生物學、基因組學、醫學等知識,確認影響發病的基因;或是藉由比對健康雙親的基因,從可能的序列中搜尋基因變異處,再找出罕見遺傳疾病的發病機制。

接著,再利用「全基因體圖譜繪製」,來組裝長片段的 DNA,以提高基因排列的正確性、以及偵測結構性變異的靈敏度。

全基因體圖譜繪製流程。資料來源│Vilella Genomics  圖片重製│張語辰

如上圖所示,郭沛恩團隊建立基因圖譜 (gene map) 的方法,是先將 DNA 分成較大的片段,利用特定的酵素來標記 DNA 上的特定序列,並黏上螢光當作標籤 (label)。透過機器測量各個螢光標籤之間的距離後,再從影像組合成長 DNA 片段,並定位出該 DNA 片段在染色體的位置。

全基因體圖譜繪製,彌補了全基因體定序讀取長度限制的缺憾;而全基因體定序,則提供了準確的 DNA 序列。藉由這兩個方式,團隊得以進行全基因組遺傳分析 (full genome analysis) ,進而探討罕見遺傳疾病的致病因子。郭沛恩提到:

「DNA 裡充滿重複的序列,讓重建工程增添難度。但如果找到特殊的序列,對定序會很有幫助。」。

相較於小片段 DNA,團隊所使用的較大 DNA 片段有更多序列線索,可以辨認、拼湊出基因原本排列的順序。將辨認大片段 DNA 得到的基因圖譜拼在一起,就能重組整條染色體。

圖/wikipedia

大眾常見疾病:分析多基因關聯

對於「單一」基因異常造成的罕見遺傳疾病,能藉由全基因組定序 (whole Genome Sequencing) 和全基因組圖譜繪製 (whole genome mapping) ,找到可能致病的遺傳因子,再進一步研究發病原因與機制。

但面對「多基因」交互影響的疾病,例如糖尿病、癌症、心血管疾病、精神疾病等等,上述的全基因組遺傳分析 (full genome analysis) 就無法測出各個單一基因對疾病的影響程度。因為,以心血管疾病來說,少部分為單基因遺傳疾病,而大部分為多基因加上環境飲食等因子交互影響所致,病因相當複雜。

圖/maxpixel

針對多基因影響的常見疾病,郭沛恩研究團隊利用另一種方法:全基因組關聯分析 (genome-wide association study,簡稱 GWAS )。

全基因組關聯分析 (GWAS) 搭配醫療資料的大數據分析,期望能找到與疾病預防、進程、及治療相關的生物標記 (biomarker),也就是體內可以反應某種疾病狀態的指標。目前,郭沛恩團隊正積極地與臺灣各大醫院合作,運用此方法,推行和大眾健康息息相關的「臺灣精準醫療計畫」。

在臺灣精準醫療計畫中,郭沛恩團隊透過文獻收集與疾病相關的「已知」基因變異點,並設計適合檢測該基因位點的晶片,之後病人的檢體就會利用這張晶片進行全基因組關聯分析 (GWAS),確認病人的基因是否在該處有變異情況。

另一方面,由於多數疾病的基因變異點尚「未知」,團隊可藉由全基因組關聯分析 (GWAS) 探究大量的病患基因樣本,從大數據找出與該疾病相關的致病基因;換句話說,當某種疾病的大多數病患,幾乎都在某處基因出現問題,就能推測其為關鍵的基因變異位點。

若想了解個人罹患糖尿病、癌症、心血管疾病等等這類常見疾病的風險,大眾可先進行基因檢測,建立自己的基因遺傳輪廓 (gene profile),並透過上述的全基因組關聯分析 (GWAS) 觀察基因變異點或表現量,再與自己的臨床病歷比對分析,就可從基因評估患病風險,比參考家族病史更為準確。

圖/wikipedia

降低藥害風險:體外基因檢測

由於不同的基因型會影響人體對藥物的反應,經由體外的基因檢測 (gene test),也能事先得知病患對藥物的反應,協助醫生選用最適合的藥物。基因檢測除了讓醫療決策更精準,用藥也能更符合個人需求。目前市面流通的藥物,多數是由歐美國家針對西方人開發,用在亞洲人身上可能沒有效果、甚至有害。例如,治療心血管疾病常用的抗凝血劑 Warfarin (音譯華法林)。

Warfarin 是從老鼠藥研發而來,具有相當程度的毒性,運用於人體醫療可以預防中風、心臟病。但因為亞洲人缺乏代謝此藥物的基因,很容易產生副作用,用藥過量會出血死亡。2006 年,中研院陳垣崇院士已經找到 Warfarin 的基因標的,並與醫院合作進行用藥前的基因篩檢。

至於常用於治療癲癇與三叉神經痛的藥物 Carbamazepine (卡巴氮平),誤用會引起史蒂文生氏強生症候群 (SJS), 與毒性表皮溶解症 (TEN) 等皮膚病徵。在用藥前先進行基因檢測,可以避免病人為副作用所困擾。

圖/flickr

目前在臺灣部分大醫院進行單一位置的基因檢測,所費不貲。後續郭沛恩研究團隊若與健保合作,可以讓大眾用較低的費用了解自己的基因組資訊。

但為了達成精準醫療,除了檢測個人基因資訊,還需要蒐集臨床病歷資料、日常運動與飲食習慣等等。資訊越詳細,對疾病預測、治療越有幫助,但前提是要獲得個人的同意。郭沛恩想像,或許有一天,人人身上都有穿戴式裝置的追蹤器,就可以精確蒐集各種訊息。

「但個人意願如何,這就不知道了。」郭沛恩說。

需要時間證明成效

圖/pexels

2018 年初,針對罕見遺傳疾病的全基因組遺傳分析 (full genome analysis) 計畫才剛開始,至少需要一年的時間來執行;至於目標是運用全基因組關聯分析 (genome-wide association study) 提升大眾健康的「臺灣精準醫療計畫」,郭沛恩則期望在這個計畫推行一年半的時候,評估與健保系統合作的可行性,以及是否能真的找到目前未知的致病基因。

與疾病篩檢的「目的性」檢驗不同,臺灣精準醫療計畫偏向「預防性」檢測,計畫推廣的同時,需要更大量的樣本協助研究。但基因組分析通常會是在人們健康的時候進行,不同於以往大眾所熟悉的目的性疾病篩檢,推廣的過程勢必會受到質疑。面臨這個挑戰,郭沛恩表示研究團隊必須加強示範基因檢測的實際益處,而非以純粹提供數據讓科學家研究為理由來說服民眾。

可預先分析新生兒的基因嗎?

圖/pexels

分析雙親的基因組資訊,的確可以預測新生兒可能面臨的遺傳疾病,但實際狀況依然要等胚胎成形後才能確認。若是從胚胎中取出細胞,並將細胞核中的基因組完全定序,避開疾病與缺陷,只留下優良的基因組合就不再只是夢想。

要完整分析基因組,需要約一百萬個細胞。但在胚胎時期,頂多只能取得幾個細胞,遠不及基因定序所需的量。但即使技術上可行,在倫理層面,郭沛恩還是覺得極不妥當。「我們不知道甚麼樣的寶寶是最好的。」郭沛恩認為,聰明的、健康的、外貌出色的,對每位父母而言「最好的寶寶」都不一樣。

況且,不符合這些特徵的新生兒,並不代表沒有活下來的權力。因此郭沛恩強調,用全基因組分析來篩選小孩,絕對不是個好主意。

臺灣得天獨厚的優勢

圖/wikipedia

時任生醫所所長的劉扶東升任中研院副院長後,原本一年約莫來臺灣一次的郭沛恩收到擔任所長的邀請。但對於香港長大,在美國就讀大學,並一路當上加州大學舊金山分校皮膚科教授、心血管研究所特聘教授的郭沛恩來說,離開熟悉的環境需付出相當龐大的代價。「我其實沒有想過要離開舊金山,因為研究進行得很順利。」

不過,得知有機會能將精準醫療計畫付諸實行後,郭沛恩對來到臺灣工作相當期待。因為歐美國家也曾推行精準醫療,但歐洲國家如瑞典、荷蘭,將重點放在費用昂貴的全基因體定序。「一個人定序要一千美金,瑞典全國定序要花一百億,很難找到這麼多經費。」郭沛恩提到執行的困難時說。

至於美國,除了民族組成複雜增加分析難度,缺乏完善的健康福利政策更讓基因醫療窒礙難行。由於美國人民的健康保險,是由任職的公司向合作的私人保險集團投保,只要換工作就會由新的保險公司接手,因此當下發生意外或疾病才會給付醫療服務。加上私人保險集團以營利為宗旨,防患未然的基因檢測很難要求保險公司給付。

有些國家有健康照護問題,有些國家則是樣本數量不足。擁有全民健保、人口又夠多的臺灣,確實是個合適的地方。「再說,政府也很有興趣,因為這計畫有機會節省醫療支出,又讓大家更健康。」郭沛恩說。

在美國進行研究多年,後應邀來臺灣,熟悉兩方研究環境的郭沛恩指出,兩國在研究領域都有聰明的人才,差異在於行政系統。在美國,每一筆花費都必須各別申請,雖然申請很困難,但獲得的經費很多;至於臺灣,整筆研究經費是來自國家,雖然資源有其侷限,但研究員有較大的自主空間,可以探索感興趣的領域。

和能力相應的薪資與友善的環境,是招募人才不變的道理

圖/flickr

對於人才招募,郭沛恩認為:

資源(或薪資)、名譽、還有工作的快樂,是讓人有動力工作的要素。

只要臺灣能提供與能力相對應的待遇,就能吸引各地優秀的人才一起來合作。問及現在工作的心得,郭沛恩笑著說:「這裡的同事彼此會熱心地提供協助,工作起來很愉快。」

延伸閱讀

本著作由研之有物製作,原文為《破解遺傳疾病、評估大眾用藥風險──全基因組分析》以創用CC 姓名標示–非商業性–禁止改作 4.0 國際 授權條款釋出。

小朋友暑假不上課要幹嘛?當然是要一直玩一直玩啊~

但是玩樂也要有收穫

在這個充滿無限可能性的「加速時代」

就算不能預測未來、也要讓您家的小朋友擁有跟上未來的「自主學習力」

該怎麼獲得自主學習力呢? 2018 泛科夏令營幫您的小朋友準備好了!

 

 

The post 全基因組分析能讓我們知道多少事? appeared first on PanSci 泛科學.

from PanSci泛科学:http://pansci.tw

145 对 “全基因組分析能讓我們知道多少事?”的想法;

  1. Does your website have a contact page? I’m having a tough time locating it but, I’d like to shoot you an email. I’ve got some creative ideas for your blog you might be interested in hearing. Either way, great blog and I look forward to seeing it expand over time.|

  2. Hi there! I know this is kinda off topic nevertheless I’d figured I’d ask. Would you be interested in trading links or maybe guest authoring a blog article or vice-versa? My site discusses a lot of the same topics as yours and I believe we could greatly benefit from each other. If you happen to be interested feel free to send me an e-mail. I look forward to hearing from you! Superb blog by the way!|

  3. I was wondering if you ever considered changing the structure of your website? Its very well written; I love what youve got to say. But maybe you could a little more in the way of content so people could connect with it better. Youve got an awful lot of text for only having 1 or two pictures. Maybe you could space it out better?|

  4. I’m more than happy to uncover this web site. I wanted to thank you for ones time just for this fantastic read!! I definitely savored every little bit of it and I have you book-marked to look at new things in your web site.|

  5. When I initially commented I appear to have clicked the -Notify me when new comments are added- checkbox and now every time a comment is added I receive four emails with the exact same comment. Perhaps there is a way you can remove me from that service? Kudos!|

  6. Hello There. I discovered your blog the use of msn. This is a very neatly written article. I’ll be sure to bookmark it and return to read extra of your useful info. Thanks for the post. I will certainly comeback.|

  7. Hello! This post couldn’t be written any better! Reading this post reminds me of my previous room mate! He always kept talking about this. I will forward this write-up to him. Fairly certain he will have a good read. Many thanks for sharing!|

  8. Excellent goods from you, man. I have understand your stuff previous to and you’re just too excellent. I actually like what you have acquired here, really like what you’re saying and the way in which you say it. You make it entertaining and you still take care of to keep it sensible. I can’t wait to read much more from you. This is really a terrific web site.|

  9. I absolutely love your website.. Great colors & theme. Did you make this website yourself? Please reply back as I’m attempting to create my own site and would love to find out where you got this from or just what the theme is called. Appreciate it!|

  10. I simply want to mention I’m new to weblog and honestly liked you’re web site. Likely I’m likely to bookmark your blog . You actually have wonderful articles. Kudos for revealing your web-site.

  11. I have been surfing online more than 2 hours today, yet I never found any interesting article like yours. It’s pretty worth enough for me. Personally, if all website owners and bloggers made good content as you did, the web will be much more useful than ever before.|

  12. Hey there great website! Does running a blog like this take a massive amount work? I have absolutely no knowledge of coding however I was hoping to start my own blog soon. Anyhow, should you have any recommendations or tips for new blog owners please share. I know this is off subject nevertheless I simply needed to ask. Kudos!|

  13. I’ll right away take hold of your rss feed as I can not in finding your email subscription hyperlink or newsletter service. Do you’ve any? Kindly let me know so that I could subscribe. Thanks.|

  14. Wonderful blog! I found it while surfing around on Yahoo News. Do you have any tips on how to get listed in Yahoo News? I’ve been trying for a while but I never seem to get there! Appreciate it|

  15. Hey there this is kind of of off topic but I was wondering if blogs use WYSIWYG editors or if you have to manually code with HTML. I’m starting a blog soon but have no coding expertise so I wanted to get advice from someone with experience. Any help would be greatly appreciated!|

  16. I’m impressed, I must say. Rarely do I encounter a blog that’s both educative and interesting, and without a doubt, you have hit the nail on the head. The problem is something that too few folks are speaking intelligently about. I’m very happy I stumbled across this in my hunt for something concerning this.|

  17. Sweet blog! I found it while surfing around on Yahoo News. Do you have any suggestions on how to get listed in Yahoo News? I’ve been trying for a while but I never seem to get there! Thanks|

  18. Hello, Neat post. There is an issue together with your site in internet explorer, would test this? IE still is the marketplace chief and a good section of people will omit your wonderful writing due to this problem.|

  19. I would like to express my appreciation to you just for bailing me out of this setting. Just after researching through the internet and seeing basics which were not pleasant, I thought my entire life was done. Living minus the solutions to the problems you’ve fixed through your entire short post is a serious case, as well as ones that might have in a wrong way damaged my entire career if I hadn’t come across your web page. The training and kindness in maneuvering every item was very helpful. I am not sure what I would have done if I hadn’t encountered such a point like this. I can at this point relish my future. Thanks so much for your high quality and result oriented help. I won’t hesitate to suggest your site to any person who requires guide on this subject.

  20. Howdy very nice blog!! Man .. Beautiful .. Superb .. I will bookmark your site and take the feeds also¡KI am glad to search out numerous useful information here in the publish, we’d like develop extra strategies on this regard, thanks for sharing. . . . . .

  21. I would like to thank you for the efforts you’ve put in writing this website. I am hoping the same high-grade site post from you in the upcoming also. Actually your creative writing abilities has inspired me to get my own website now. Actually the blogging is spreading its wings rapidly. Your write up is a good example of it.

  22. Nice post. I was checking constantly this blog and I am impressed! Very helpful information particularly the last part 🙂 I care for such information much. I was seeking this particular information for a long time. Thank you and good luck.

  23. Somebody essentially assist to make seriously posts I would state. That is the first time I frequented your website page and up to now? I amazed with the research you made to make this actual post extraordinary. Great job!

  24. hi!,I like your writing so so much! proportion we communicate extra about your article on AOL? I need an expert on this house to resolve my problem. May be that’s you! Having a look ahead to peer you.

  25. I wanted to construct a small word to say thanks to you for those fantastic secrets you are placing at this website. My particularly long internet look up has finally been honored with wonderful suggestions to talk about with my family. I would assume that many of us site visitors are very much endowed to exist in a useful network with many special individuals with great techniques. I feel pretty grateful to have seen your webpages and look forward to many more fabulous moments reading here. Thanks once again for everything.

  26. I and my buddies appeared to be reviewing the good information and facts from your web site then then I got an awful feeling I had not expressed respect to the web site owner for those secrets. All of the guys appeared to be consequently very interested to study them and now have simply been taking pleasure in these things. Appreciate your indeed being well kind as well as for figuring out such beneficial themes millions of individuals are really wanting to understand about. My personal honest regret for not saying thanks to you earlier.

  27. I do agree with all the ideas you have offered in your post. They are really convincing and can definitely work. Nonetheless, the posts are very short for starters. May just you please prolong them a little from subsequent time? Thank you for the post.

  28. A person necessarily lend a hand to make significantly articles I might state. This is the first time I frequented your web page and up to now? I amazed with the research you made to make this particular post incredible. Excellent process!

  29. Somebody essentially assist to make significantly posts I might state. This is the first time I frequented your web page and so far? I surprised with the research you made to create this actual publish incredible. Wonderful process!

  30. Someone essentially lend a hand to make critically articles I might state. This is the first time I frequented your website page and to this point? I amazed with the analysis you made to make this particular publish incredible. Wonderful task!

  31. I¡¦m now not positive where you are getting your info, however good topic. I must spend a while finding out more or understanding more. Thank you for magnificent information I used to be searching for this information for my mission.

  32. Definitely believe that which you said. Your favorite justification appeared to be on the internet the easiest thing to be aware of. I say to you, I definitely get annoyed while people think about worries that they just don’t know about. You managed to hit the nail upon the top and also defined out the whole thing without having side-effects , people could take a signal. Will likely be back to get more. Thanks

  33. Great ¡V I should certainly pronounce, impressed with your site. I had no trouble navigating through all tabs as well as related info ended up being truly simple to do to access. I recently found what I hoped for before you know it at all. Reasonably unusual. Is likely to appreciate it for those who add forums or something, site theme . a tones way for your client to communicate. Nice task..

  34. I intended to draft you that very small note to help say thanks once again with your stunning thoughts you’ve documented at this time. It has been quite pretty generous with you to present openly all that many of us would have sold as an e-book to get some money for themselves, chiefly now that you might well have done it if you wanted. Those good ideas as well served like the easy way to be aware that other people online have the identical eagerness like my own to grasp significantly more in regard to this matter. I know there are some more pleasant periods in the future for folks who check out your site.

  35. I do consider all of the concepts you’ve introduced in your post. They’re very convincing and will definitely work. Nonetheless, the posts are very short for starters. May you please extend them a little from next time? Thank you for the post.

  36. Good ¡V I should definitely pronounce, impressed with your site. I had no trouble navigating through all the tabs and related info ended up being truly easy to do to access. I recently found what I hoped for before you know it in the least. Reasonably unusual. Is likely to appreciate it for those who add forums or something, site theme . a tones way for your client to communicate. Excellent task..

  37. I am also commenting to make you know what a beneficial discovery my friend’s child undergone checking your site. She came to understand a good number of details, most notably what it’s like to have an awesome coaching nature to make men and women with ease fully grasp a number of advanced matters. You actually surpassed our expected results. Thank you for churning out those informative, dependable, revealing as well as unique thoughts on the topic to Kate.

  38. I like the helpful info you provide in your articles. I will bookmark your blog and check again here frequently. I’m quite sure I will learn a lot of new stuff right here! Good luck for the next!

  39. I’m not sure where you’re getting your info, but great topic. I needs to spend some time learning much more or understanding more. Thanks for fantastic info I was looking for this info for my mission.

  40. This is very interesting, You’re a very skilled blogger. I’ve joined your rss feed and look forward to seeking more of your excellent post. Also, I have shared your web site in my social networks!

  41. I’m extremely impressed with your writing skills as well as with the layout on your blog. Is this a paid theme or did you modify it yourself? Anyway keep up the excellent quality writing, it is rare to see a great blog like this one today..

  42. Excellent blog here! Also your web site loads up fast! What web host are you using? Can I get your affiliate link to your host? I wish my website loaded up as fast as yours lol

  43. Excellent blog right here! Also your web site so much up very fast! What host are you using? Can I am getting your associate hyperlink in your host? I wish my website loaded up as fast as yours lol

  44. Wonderful work! This is the kind of information that are meant to be shared across the internet. Disgrace on the seek engines for now not positioning this submit upper! Come on over and discuss with my web site . Thank you =)

  45. I would like to thank you for the efforts you have put in writing this site. I am hoping the same high-grade web site post from you in the upcoming also. Actually your creative writing skills has inspired me to get my own blog now. Really the blogging is spreading its wings fast. Your write up is a good example of it.

  46. I simply wanted to appreciate you once more. I do not know the things I would’ve tried without the tips provided by you regarding such a industry. It was before a horrifying setting in my circumstances, however , seeing your specialized manner you dealt with the issue made me to weep with delight. I am just grateful for your work and in addition believe you know what a great job you have been doing educating most people through the use of a site. I know that you have never come across all of us.

  47. Pretty section of content. I just stumbled upon your web site and in accession capital to assert that I get in fact enjoyed account your blog posts. Any way I will be subscribing to your augment and even I achievement you access consistently rapidly.

  48. of course like your web-site however you need to take a look at the spelling on quite a few of your posts. Several of them are rife with spelling problems and I to find it very bothersome to tell the reality nevertheless I¡¦ll definitely come back again.

  49. Thanks so much for giving everyone remarkably superb possiblity to read critical reviews from this website. It’s always very excellent and also full of a lot of fun for me and my office acquaintances to search your website nearly three times in one week to find out the latest guides you have got. Not to mention, I’m at all times fulfilled considering the breathtaking techniques you give. Selected two areas in this article are rather the most beneficial I’ve ever had.

  50. I just could not go away your web site before suggesting that I actually enjoyed the usual information a person supply to your guests? Is gonna be again often in order to investigate cross-check new posts

  51. It¡¦s in reality a nice and helpful piece of information. I¡¦m glad that you shared this helpful information with us. Please keep us informed like this. Thank you for sharing.

  52. I¡¦ve been exploring for a bit for any high-quality articles or blog posts on this kind of house . Exploring in Yahoo I ultimately stumbled upon this web site. Reading this info So i am happy to convey that I have a very just right uncanny feeling I found out just what I needed. I so much indubitably will make certain to do not disregard this web site and provides it a look regularly.

  53. I cling on to listening to the news update talk about getting boundless online grant applications so I have been looking around for the most excellent site to get one. Could you tell me please, where could i get some?

  54. What i don’t understood is in truth how you’re no longer actually much more neatly-appreciated than you may be right now. You’re very intelligent. You already know thus significantly on the subject of this topic, produced me personally consider it from a lot of varied angles. Its like women and men are not involved except it is something to accomplish with Woman gaga! Your personal stuffs great. Always take care of it up!

  55. I must express my love for your generosity supporting persons that really want help on in this topic. Your very own commitment to passing the solution throughout turned out to be amazingly helpful and have frequently permitted others like me to get to their endeavors. The warm and helpful report signifies so much to me and additionally to my colleagues. Thank you; from each one of us.

  56. Hello There. I found your blog using msn. This is an extremely well written article. I will make sure to bookmark it and return to read more of your useful information. Thanks for the post. I will definitely comeback.

  57. Good web site! I really love how it is simple on my eyes and the data are well written. I am wondering how I might be notified whenever a new post has been made. I’ve subscribed to your feed which must do the trick! Have a great day!

  58. Attractive section of content. I just stumbled upon your weblog and in accession capital to assert that I acquire actually enjoyed account your blog posts. Anyway I will be subscribing to your augment and even I achievement you access consistently rapidly.

  59. hi!,I love your writing so a lot! proportion we be in contact more about your article on AOL? I need a specialist on this house to solve my problem. May be that is you! Looking ahead to see you.

  60. Hiya, I am really glad I’ve found this info. Nowadays bloggers publish just about gossips and web and this is actually frustrating. A good site with interesting content, that’s what I need. Thank you for keeping this site, I’ll be visiting it. Do you do newsletters? Can not find it.

  61. I enjoy you because of all of your labor on this web site. Betty take interest in going through internet research and it is obvious why. Most people notice all regarding the powerful form you produce very important guides by means of your web site and in addition improve contribution from people about this issue then our favorite simple princess is actually being taught a great deal. Take advantage of the remaining portion of the year. You’re carrying out a really good job.

发表评论

电子邮件地址不会被公开。 必填项已用*标注