EBológia : Buksi és Kedves - A kutyamatuzsálemek különleges génjei

English below

Buksi (balra) és kedves (jobbra, Sándor Sára genetikussal)

Matuzsálemi, 22 és 27 éves kort megélt kutyák vizsgálatával olyan génváltozatokat azonosítottak az ELTE kutatói, amelyek eddigi ismereteink szerint nem fordulnak elő átlagos élethosszú kutyákban.

Minél tovább él valaki, annál nagyobb a genetikai tényezők súlya az öregedésben. A legöregebb matuzsálemek genomja halmozottan tartalmaz az élettartamot növelő génváltozatokat. A legtöbb ilyen gén a sejtek energiatermelésével, önfenntartásával, a DNS-hibák javításával kapcsolatos funkciókat lát el. E gének azonosításához általában nagyon nagy mennyiségű adat elemzésére van szükség, minek során kapcsolatot keresnek az átlagnál hosszabb életkor és egyes génvariánsok előfordulása között. Az ELTE Etológia Tanszék Szenior Családi Kutya Programjának új cikke különlegesen hosszú életű kutyák élethosszáért felelőssé tehető genetikai variánsokat azonosított. A tanulmány különlegességét az adja, hogy a genetikai analízis két, matuzsálem életkorú kutya teljes genomi információján alapult, amelyeket 850, átlagos élethosszú kutyáéval vetettek össze. Az eredmények fontos kezdeti lépést jelenthetnek a kutyák egészséges öregedésének és hosszú életének genetikai feltételeit feltáró kutatások számára. A tanulmány két matuzsálem korú alanya már ismerős lehet a: Buksi, a sárrétudvari Pegazus lovardában 27 évet élt keverék kan és Kedves, az ócsai Szent Ferenc Állatotthon lakójaként egészségben megöregedett 22 éves keverék szuka igazi hírességgé vált. A kutatók is ennek köszönhetően értesültek a két különleges ebről és mintát vettek az állatoktól. A genetikai kutatásokhoz többnyire elegendő a kutyák szájnyálkahártyájáról vett törlet, így a mintavétel egy kis csiklandozáson kívül nem jelent semmilyen kellemetlenséget számukra. Gazdáik beleegyezése után, mivel különleges helyzetről volt szó, Buksi és Kedves állatorvosi segítséggel ezen kívül pár csepp vérrel is támogatta a tudományt – a speciális eljárás, az úgynevezett „teljesgenom-szekvenálás” ugyanis érzékeny technika, és legmegbízhatóbban vérből izolált DNS-ből kivitelezhető.

Sajnos Kedves és Buksi már nem él, de egészen egyedülálló hagyaték maradt utánuk, mivel a mintáikból sikerült teljes genomi információjukat kinyerni a szekvenálás segítségével. Egy kutya genomja körülbelül 2,5 milliárd genetikai betűt - ún. bázist - tartalmaz, ráadásul minden betűt duplán, annak megfelelően, hogy minden testi kromoszómából (ez a DNS becsomagolt formája) két példánnyal rendelkezik. Ennek az összesen mintegy 5 milliárd betűnek a megfelelő sorrendben való kiolvasása és összerendezése hatalmas számítástechnikai kapacitást igényel. Egyetlen, a nyers adatokat tartalmazó fájl száznál is több gigabájt helyet foglalhat egy számítógép merevlemezén.

DNS-gyűjtés Buksitól

“Az adatokat szuperszámítógépek használatával elemeztük ki, azt keresve, hogy milyen genetikai variánsok fordulnak elő a két matuzsálem kutyában, és ezek mutatnak-e egymással átfedést.” - mondta el Jónás Dávid bioinformatikus, a Frontiers in Genetics folyóiratban megjelent tanulmány első szerzője. A legígéretesebb gének közül 4 a génkifejeződés szabályozásával, ezáltal pedig a sejtek alapvető funkcióinak finomhangolásával hozható kapcsolatba, ami egybevág a legújabb szakirodalmi eredményekkel, és egy izgalmas kutatási irányt vetít előre.

Az analízist egy korábbi, emberi matuzsálemeket vizsgáló kutatás mintájára is lefuttatták, célzottan az öregedéssel összefüggésbe hozható génekre fókuszálva. A két kutya esetében 24 ilyen variánst sikerült felderíteni.

“Amikor a kutyák genomjában található mind a húszezer gént megvizsgáltuk, néhány génben jelentős, úgynevezett start- és stop-kodon mutációkat találtunk. Ezeket érdemes volna tovább vizsgálni, első körben igazolva azt, hogy valós mutációkról van szó, majd pedig feltárni a biológiai hatásaikat is” - javasolja Sándor Sára, genetikus.

Buski és a kutatók

Kubinyi Enikő etológus, a kutatás vezetője arra hívja fel a figyelmet, hogy a különlegesen hosszú életkor elérésében a környezet szerepe is fontos. “Buksit és Kedvest is meglátogattuk (videó lent vagy itt). Mindketten vidéken, teljesen szabadon éltek és sok kutyával, emberrel voltak kapcsolatban. Ebben hasonlítottak a 29,5 évet élt világcsúcstartó ausztrál matuzsálemhez, Bluey-hez. Táplálkozásukat tekintve viszont különbözött a helyzet. Buksit egész életében nyers csirkével és háztáji maradékkal etették, fiatalabb korában ürgékre és pockokra is vadászott a pusztán, míg Kedves ugyanazt ette, amit az ócsai állatotthon többi lakója.”

A kutatók szeretnék eredményeiket megerősíteni további idős kutyák vizsgálatával, ezért kérik, hogy aki 22 éven felüli kutyát ismer és szívesen részt venne a kutatásban, jelentkezzen a honlapjukon vagy ezen a címen: seniorfamilydogproject@gmail.com.

Referencia: Jónás, D., Sándor, S., Tátrai K., Egyed, B., Kubinyi, E. (2020) A preliminary study to investigate the genetic background of longevity based on whole-genome sequence data of two Methuselah dogs. Frontiers in Genetics, 11:315. doi: 10.3389/fgene.2020.00315

Unique genes of canine “centenarians”

New research identified gene variants unique to two Methuselah dogs (aged 22 and 27 years) which could not be found in 850 dogs of average age. The genome of the oldest old “Methuselahs” is enriched with gene variants that can elongate lifespan.

In humans, the age of 100 years is considered special by most people. Indeed, in terms of the human lifespan, centenarians live at least 40-70% longer than the average. The corresponding age in dogs would be 17-22 years, depending on the breed, making these Methuselah dogs an exceptional model for the study of aging and longevity.

The longer an individual lives the greater the impact genetics has on its lifespan. The genome of the oldest old “methuselahs” is enriched with gene variants that can elongate lifespan and, most importantly, according to studies, contains fewer variants that can predispose the individuals to certain diseases. Most longevity genes have functions linked to cellular homeostasis, energy production, and DNA damage repair mechanisms. The identification of such genes usually requires a huge amount of data to be analyzed to search for connections between the longer lifespan of individuals and the occurrence of certain gene variants in a population. A recent paper from the Senior Family Dog Project at the Eötvös Loránd University, Hungary, has reported the identification of gene variants that could be linked to the extreme old age of some dogs. The uniqueness of the genetic analysis done in this study lies in the applied method: the researchers sequenced, analyzed and made publicly available the full genome of the two Methuselah dogs. The results could represent a main first step to open up new directions for investigations that search for the key factors behind healthy aging and longevity in dogs.

Buksi, an intact male mixed-breed lived 27 years in a farm in Sárrétudvari (Pegazus Lovarda, Hungary), and Kedves, a spayed female mixed-breed lived 22 years in an animal shelter at Ócsa (Szent Ferenc Animal Shelter Foundation; Hungary). The researchers have become aware of the two extremely old dogs thanks to local veterinarians and shelter volunteers who wanted to introduce these really special dogs to a wider audience through the media. Usually, genetic investigations can be performed from buccal swabs, which are easy to collect from the animals and won’t cause any inconvenience – apart from a minor tickling. However, the researchers wanted to make sure that they can unfold the full potential their genome could hold, so they also collected a small amount of blood with the consent of the owners and with the help of experienced veterinarians. Blood is a reliable source of genomic DNA and could be superior to buccal swabs in quality, when high-throughput genomic techniques, like genome-sequencing, are to be used.

Sadly, both Kedves (†22) and Buksi (†27) have already passed away, but they left a truly unique legacy, since their complete DNA was extracted from their samples and then sequenced by the scientists. The genome of the dog contains approximately 2.5 billion genetic letters (so-called base pairs) – and every one of them twice, because each chromosome (i. e. the compact form of the DNA) is present twice in every individual, save the sex chromosome of males, who have X-Y chromosomes, instead of the X-X chromosomes of females. This results in a total of 5 billion genetic letters per individual, and its reading and the complete reconstruction of the genetic code requires enormous computational capacities. One file containing the raw sequence data of a single individual requires more than 100 GB of storage space on any computer’s hard drive.

“We analyzed this data using one of the Hungarian supercomputers, searching for the gene variants in the Methuselah dogs and investigating the overlaps of gene variants between the 2 individuals.” – said Dávid Jónás bioinformatician, the first author of the article published in the Frontiers in Genetics journal. Four out of the most promising genes were related to the regulation of gene expression and therefore might be related to the fine-tuning of basic cellular functions, which is in line with the most recent scientific findings related to aging and establishes an interesting direction for future research. Another analysis, based on a study of human centenarians, found 24 gene variants, which are specifically hypothesized to be related to aging and longevity.

“When we examined the approximately twenty thousand canine genes, we found some so-called start- and stop-codon mutations, which might result in disrupted gene functions. It is worth further examining these genes: first to verify that these mutations are true positives, then to investigate their biological effects as well” – said Sára Sándor, a geneticist.

Ethologist Eniko Kubinyi, the leading scientist of the research said that the environment has also a very important role in the development of an extremely long lifespan. “We visited both Buksi and Kedves (video is available here). Both of them lived in the countryside, they could roam freely and both of them had contact with a lot of other dogs and humans alike. In this regard, they were similar to the world-recorder dog, Bluey (a cattle dog), who lived 29.5 years in Australia. However, the two Methuselah dogs in this study had very different diets. Buksi was fed with raw chicken and household leftover and used to hunt for small rodents in his younger age. Kedves, on the other hand, was fed with the same dog food as the other dogs at the animal shelter.”

The scientists would like to confirm their findings by analyzing more dogs of extreme age and therefore ask those owners, who have a dog aged 20 years or more and who are willing to participate in the study to contact them at their website.

Interview with David Jonas, PhD, first author of the study

What motivated you to investigate the genomes of extremely elderly dogs?
◦ Firstly, dogs are an ideal model animal to study aging and extreme longevity. Dogs share the same very complex environment with their owners to an extent no other animal does. In contrast, laboratory animals, for example, are studied in a highly controlled environment, and therefore it is not known how much the results obtained from such experiments are adaptable to humans. Studying aging directly in humans is very important, but due to the very long lifespan, this is not practical; the average age of dogs is much shorter and therefore these studies in dogs are faster.
Secondly, aging is a very complex phenomenon, influenced by both genetic and environmental factors (this latter group includes – among others: physical activity, diet, healthcare, habitat, etc.). The genetic factors, however, are of special interest, since these cannot be changed as quickly and efficiently as the environmental factors. Furthermore, Methuselah dogs are those, who aged “successfully”; a better understanding on their aging and their genetic background can help in the future to uncover the genetic background of age-related diseases, such as the canine cognitive dysfunction (which is very similar in nature to the Alzheimer disease of humans).
Finally, also in their own right, dogs are of interest to study aging. If we better understand the different factors affecting aging and healthy aging in particular, we can promote the appropriate lifestyle for dog owners. If the pet dogs live longer and healthier, it would have a positive impact not only on the pets but on the mental health of their owners as well.
What age of dogs is considered a Methuselah dog?
◦ In human studies, usually centenarians (100+ years of age) are called “Methuselah”, although sometimes younger people (e.g. 90+ years) are also included in the studies. Considering the centenarians studied by Han et al. (2013), these elderly people lived ~50% longer than the average human lifespan – which is ~72 years according to a WHO estimate from 2018. In our article, we defined Methuselah dogs considering this increase (i.e. +50%) in lifespan and the average canine lifespan estimated by Inoue et al. (2018). This gives an “overall total estimate”: a dog would be considered Methuselah if it lived 17+ years, however, considerable variation exists due to different factors (e.g. smaller breeds or mixed-breed individuals are known to live longer, while larger breeds, in general, have a shorter lifespan). In the case of mixed-breed individuals, 22.5+ years old dogs would be considered as Methuselah dogs.
What did the research find? Are you surprised by any of the findings?
◦ The most surprising was that even with a sample size of 2 very old individuals and 850 dogs with the average lifespan, we found a connection between gene expression and extreme longevity, based on which we could formulate a hypothesis. Our aim now is to further investigate this hypothesis and validate it (please see our answer to your question “What does the study tell us that we did not already know?” for more details on this).
What do the findings mean for the average dog owner?
◦ This basic research, preliminary in nature, and therefore no solid conclusions could be drawn and we cannot formulate specific advice to the owners at this stage. One of our aims with this preliminary study was to highlight the high value of these extremely old dogs both in the scientific community and in the grand public. Given a larger sample size, we could e.g. investigate the “breed composition” of the mixed-breed Methuselah dogs based on DNA samples, which could give us information about the breeds that are capable of living such an extremely long life, which would be very interesting and informative for the owners as well.
What does it add to our understanding of aging for dogs (and possibly humans)?
◦ This was the first study that investigated the genetic background of aging in dogs using whole-genome sequencing methods. We made the complete DNA information of these dogs publicly available for both scientists and the general public, who is interested in the sequence repository of the [American] National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/).
Furthermore, we identified genes that are probably linked to extreme longevity in dogs, and based on our results, we also formulated a more general (i.e. species-independent) hypothesis regarding the genetics of aging (see our answer to your next question).
What does the study tell us that we did not already know?
◦ One of the most important results is that based on our results, we could formulate a hypothesis that a crucial genetic requirement of extreme longevity lies within the fine-tuning (i.e. the superior calibration) of gene expression. In order to provide solid evidence for this, we need a larger sample size.
Why is it important to understand how dogs age?
◦ If we better understand the mechanisms of aging in dogs, we can promote a healthier lifestyle for dogs through their owners. This would greatly increase the quality of life of both the pets and their owners. It can also contribute to better understand the aging process in humans as well, e.g. because dogs share the same environment with their owners, which presumably makes the obtained results more relevant and applicable to human studies.
Furthermore, if we better understand the process of aging in dogs that were successful in aging – i .e. the Methuselah dogs, who lived a long and healthy life –, that can provide crucial information to understand the background of age-related diseases as well, e.g. by comparing the DNA of ill dogs to those who aged successfully and identifying the differences.
Which similarities did you find in Buksi and Kedves’ DNA compared to a regular one?
o We were looking for differences when we compared their whole genome to that of 850 dogs of average age. Four out of the most promising genes unique to the two old dogs were related to the regulation of gene expression and therefore might be related to the fine-tuning of basic cellular functions, which is in line with the most recent scientific findings related to aging and establishes an interesting direction for future research. Another analysis, based on a study of human centenarians, found 24 gene variants, which are specifically hypothesized to be related to aging and longevity.
What could have contributed (diet, sociability, habitat…) to their longer lifespan?
o Aging is a complex trait, influenced by both genetic and environmental factors (and possibly an interaction between these two groups of factors as well). Diet, sociability, habitat, available healthcare, physical activity, etc. are not heritable and therefore belong to the so-called environmental factors, which are known to have a great impact on aging.
However, the study of the environmental factors influencing aging was outside the scope of this study and we did not investigate these.
What can the longevity in dogs tell us about the human one?
o Information on canine aging can help us answer some human aging-related questions, such as „How much are the results obtained from lab animals in highly controlled environments applicable to humans?”. Also, the dog as a model animal in age-related studies allows us to design experiments to investigate the effect of e.g. different nutrients or treatments on aging, which experiments would be infeasible in humans. If a large community of dogs and dog owners would participate, the investigation of the effects of certain gene variants might be also more applicable in dogs than in humans.
You are now looking for more dogs of extreme age in the world to confirm your results. Do you think they will be easy to find?
It is difficult to find other dogs of extreme age, although based on a recent Japanese study, 1 out of 1000 dogs lives up to 22-25 years, so these dogs are “out there” in sufficiently high numbers. The most challenging part is to reach these dogs and their owners; many owners who would be happy to participate in our research doesn’t know about our projects and the scientific relevance of their dogs. Therefore, we would like to reach these dogs and their owners and we are waiting for their applications! (E-mail: kubinyie@gmail.com).

· Reference: Jónás, D., Sándor, S., Tátrai K., Egyed, B., Kubinyi, E. (2020) A preliminary study to investigate the genetic background of longevity based on whole-genome sequence data of two Methuselah dogs. Frontiers in Genetics, 11:315. doi: 10.3389/fgene.2020.00315.

More pictures here: https://drive.google.com/drive/folders/11H9nLuJ1E_OuX88l-U2epoy7Zq9FzvTd?usp=sharing

Press:

2020. május 7.

Buksi és Kedves - A kutyamatuzsálemek különleges génjei

Unique genes of canine “centenarians”

Interview with David Jonas, PhD, first author of the study