RES E A R C H Open Access

Initial survey of PLA2G6 missense variant

causing neuroaxonal dystrophy in Papillon

dogs in North America and Europe

Karthik Raj and Urs Giger

Abstract

Background: An autosomal recessive, rapidly progressive degenerative neuropathy known as infantile neuroaxonal

dystrophy (NAD) was originally reported in Papillion puppies in 1995. In 2015, a causative missense variant in the

PLA2G6 gene was identified in three affected puppies. Archived samples from Papillons clinically diagnosed with

NAD prior to 2015 as well as samples obtained from 660 Papillons from North Ameri ca and Europe between 2015

and 2017 were screened for the presence of this PLA2G6 gene variant (XM_022424454.1:c.1579G > A) using a

TaqMan assay.

Results: Archived samples from affected puppies diagnosed prior to 2015 and three more recently acquired

samples from Papillons clinically affected with NAD were all homozygous for the variant. SIFT analysis predicts that

the PLA2G6 missense substitution (XP_022280162.1:p.Ala527Thr) will not be tolerated in the iPLA

β protein. Notably,

17.5% of the 660 tested Papill ons were heterozygotes, resulting in a variant allele frequency of 0.09 2 in this initial

survey. Since then, screening for NAD in Papillons by at least 10 other laboratories and data from the Health

Committee of Papillon Club of America gathered between 2017 and 2019 reveal a variant allele frequency of 0.047.

Conclusions: This survey and data from other laboratories documents the widespread presence of the PLA2G6

variant in the Papillon population in North America and Europe. Despite the apparent declining prevalence of the

PLA2G6 variant, screening of Papillons intended for breeding is still recommended to avoid inadvertent production

of puppies with infantile NAD.

Keywords: Canine, Mutations, Screening, Breeding, Ataxia

Plain English summary

Infantile neuroaxonal dystrophy (NAD) is a rare rapidly

progressive disease first reported in Papillion dogs in

1995. Clinical manifestations of ataxia, head tremor, dif-

ficulty rising, discordant gate, limb extension, paresis, in-

ability to prehend food and water, and blindness are

observed by 1–3 months of age. A specific genetic vari-

ant in PLA2G6 was recently identified in three Papillons

with NAD in Japan. We screened Papillons with a

clinical diagnosis of NAD received prior to 2015 and

samples from an additional 660 Papillons from North

America and Europe received between 2015 and 2017

for the presence of this gene variant. All samples from

Papillons clinically affected with NAD were homozygous

for the variant. Furthermore, 17.5% of all Papillons

tested were asymptomatic heterozygotes, and therefore

able to pass on the variant to their offspring. Since 2017,

an increasing number of laboratories offer NAD screen-

ing of Papillons, often together with progressive retinal

atrophy testing. Data obtained between 2017 and 19 in-

dicates a variant allele frequency of 0.047. While all

these surveys are biased, the apparently high prevalence

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* Correspondence: [email protected]

Section of Medical Genetics (PennGen Laboratories), School of Veterinary

Medicine, University of Pennsylvania, 3900 Delancey St., Philadelphia, PA

19104-6010, USA

Canine Medicine an

Raj and Giger Canine Medicine and Genetics (2020) 7:17

https://doi.org/10.1186/s40575-020-00098-4

of the PLA2G6 variant indicates that screening of Papil-

lons intended for breeding is still recommended to avoid

unintentional production of puppies with infantile NAD.

Background

Hereditary neuropathies in dogs frequently present at an

early age and are generally progressive and fatal. Such dis-

eases have been reported in numerous dog breeds (Online

Mendelian Inheritance in Animals, OMIA) [1]. Identifica-

tion of a causative mutation for these genetic diseases al-

lows for a precise diagnosis, and can provide initial insight

into therapeutic strategies that may prevent or reduce dis-

ease progression. As such, companion animals with her-

editary diseases can serve as translational large animal

models to investigate the potential safety and efficacy of

novel treatments for human disease. However, screening

and informed breeding are pivotal to reduce the wide-

spread occurrence of disease-associated allelic variants in

future generations of companion dogs.

Neuroaxonal dystrophy (NAD) represents a group of auto-

somal recessive degenerative neuropathies characterized

histopathologically by ‘spher oids’ in the central nervous sys-

tem and caused by one of a few dysfunctional genes in

humans [2] and various animal species including dogs [1, 3].

In humans, variants in the phospholipase A2 group VI

(PLA2G6) gene, which encodes a calcium-independ ent en-

zyme essential for membrane integrity, cause PLA2G6-asso-

ciated neurodegeneration (PLAN) [4, 5].

An infantile NAD (OMIA #: 002105–9615) was first

identified in Papillion dogs in 1995 [6–11]. Clinical man-

ifestations of ataxia, head tremor, difficulty rising, dis-

cordant gate, limb extension, paresis, inability to

prehend food and water, and blindness are observed by

1–3 months of age. Axonal spheroids are found through-

out the central nervous system but not in any peripheral

nerves, and no iron accumulation was noted [7, 8, 11].

Because clinical signs are rapid and progressive, natural

death occurs at a few months of age if not prece ded by

humane euthanasia. In the absence of a genetic test to

identify carriers of this disease, breeders have excluded

any Papillon parents and littermates of affected puppies

from breeding to reduce the risk of producing affected

puppies. Recently, a homozygous missense variant (XM_

022424454.1:c.1579G > A) in PLA2G6 gene was identi-

fied in three Papillons with NAD in Japan [11]. In this

report, we tested archived samples from Papillons with a

clinicopathological diagnosis of NAD for the presence of

the PLA2G6 missense variant and provide results of an

initial genotyping survey of Papillons from North Amer-

ica and Europe.

Materials and methods

The Section of Medical Genetics and PennGen Labora-

tories had stored samples from four Papillons clinically

diagnosed with NAD prior to 2015. These samples, as

well as samples from their relatives, were analyzed for

the published PLA2G6 missense variant [11]. Based

upon the results, PennGen started to offer a genotyping

assay for the PLA2G6 variant in late 2015. Either cheek

swab or EDTA blood samples were accepted for geno-

typing. Genomic DNA was extracted using QIAamp

Blood Mini Kit (Qiagen, Hilden, Germany). A TaqMan

genotyping assay, as previously described [11], was used

to determine the genotypes.

Results and discussion

The TaqMan genotyping assay for the XM_022424454.1:

c.1579G > A variant in PLA2G6 readily differentiated the

three genotypes. The four Papillons clinically affected

with NAD were homozygous for the PLA2G6 variant,

while samples from non-affected dogs were either

homozygous for the wild-type allele or heterozygous for

the previously published pathogenic variant [11].

Between October 2015 and December 2017, samples

from 660 Papillons were received from North America

and Europe (Table 1). Only three puppies homozygous

for the pathogenic variant were found, with one each

from USA, Canada, and Europe. All three Papillon pup-

pies were clinically affected. The low number of af-

fecteds is likely due to the fact that Papillon breeders are

well aware of the typical clinical signs of NAD and may

not feel the need to submit a sample for testing to con-

firm their presumptive diagnosis. Among the 660 dogs,

17.5% were heterozygous.

When the genotype screening test became available in

late 2015, the demand was large and many breeders were

interested in knowing if their Papillon was a carrier. In-

deed, 79.5% of all requests indicated breeding and/or gen-

eral screening as the reason for testing. Following

screening, breeders were immediately able to avoid produ-

cing affecteds by mating clear (wild type; GG) to clear or

clear to carrier (heterozygote; GA). While biased, the abil-

ity to undertake more selective breeding appeared to re-

sult in a decline in variant allele frequency within the first

3 years of screening (Table 2). As such, screening can help

reduce the proportion of carriers in the population, reduce

the need for future testing, and permit the breeding of

many Papillons, particularly close relatives of an affected

puppy that would not have been bred in the absence of

testing in order to reduce potential disease risk.

As of late 2017, additional laboratories now offer the

test, resulting in a dramatic reduction in the number of

samples received by PennGen (only 14 Papillons with 5

carriers over the next 2 years). As of 2020, at least 10 la-

boratories worldwide (listed by the World Small Animal

Veterinary Association, a web resource on DNA tests for

hereditary diseases) offer genotyping for this variant as a

specific test and/or as part of Papillon breed panel

Raj and Giger Canine Medicine and Genetics (2020) 7:17 Page 2 of 4

testing or dog panel testing [12, 13]. A summary state-

ment gathered by the Health Committee of Papillon

Club of America indicates screening of an additio nal

2305 Papillons with a variant allele frequency of 0.047 in

2018–2019; this represents a decline of 50% over the

prior 3 years. It should be noted that both our survey

and that of the Club are biased by at least two factors.

First, interested breeders with affected and carrier Papil-

lons preferentially choose to scree n their dogs, biasing

the survey towards an increased allele frequency. Sec-

ond, as the signs of juvenile NAD [14] are easily

recognizable by breeders and clinicians, affected puppies

are likely subject to immediate humane euthanasia. As

such puppies may not be tested, the survey could be po-

tentially biased towards a decreas e in allele frequency.

Since 1995, there have been a few case reports of NAD

in Papillons from the United Kingdom [6] and thereafter

from Canada [10] and Japan [7–9, 11] suggesting a wide-

spread distribution of this devastating disease trait in the

breed. The Papillon breed has a relatively small breeding

pool, with likely common inbreeding and international

breedings. As the disease-related gene and variant were

only recently identified [11], the prior inability to screen

for this genetic disease resulted in its global dissemination.

Notably, our results suggest that the widespread availabil-

ity of screening tests for this PLA2G6 variant has resulted

in successful reduction of the prevalence of the variant al-

lele in the Papilion population.

The point variant XM_022424454.1:c.1579G > A in the

PLA2G6 gene reported to be associated with NAD in Pa-

pillons results in an amino acid subs titution of the 527th

of 806 amino acids from a highly conserved alanine (as

far back as zebrafish) to a threonine (XP_022280162.1:

p.Ala527Thr) [11]. This missense variant resides in the

serine lipase consensus sequence of the patatin domain

of the iPLA

β protein, and is not tolerated based on

SIFT analysis [15]. The overall structure and functional

domains of the iPLA

β enzyme are highly conserved be-

tween dog and human (90.8%) and among other species.

As expected for a missense variant, the iPLA

β protein was

expressed normally in the brain of Papillon puppies with

NAD, but was predicted to be severely dysfunctional [11].

Recently, the genotype-phenotype associations in hu-

man patients with NAD and PLA2G6 variants (so called

PLAN disorders) were reviewed [4, 5]. Interestingly, mis-

sense variants were also commonly reported in human

patients with PLAN. However, in humans, variants were

found throughout the gene and were not associated with

age of disease onset or clinical presentation of the four

PLAN subtypes [4, 5]. While the affected Papillons

present similarly to children with infantile NAD, an

analogous variant has not yet been identified in human

patients, although similar missense variants in the same

domain are observed [4].

In human medicine, a variant allele frequency of 0.1

(10%) is considered extremely high and is very rarely

seen, except in a few select populations for very select

variants [16]. While the survey of canine infantile

NAD presented here is biased, our findings together

with the initial report from Japan, clearly document

the worldwide distribution and high prevalence of the

variant PLA2G6 allele in the Papillon population.

Such findings are not unusual in specific dog breeds,

as common ancestry, founder effe cts, and inter-

national breeding [16] can lead to high frequencies of

a genetic disease in afflicted breeds. A common an-

cestor for the PLA2G6 gene variant has not been

identified.

Table 1 Genotyping survey results from 2015 to 2017 of Papillon dogs from various geographic regions

Region Dogs

Tested

PLA2G6 Genotypes (c.1579G > A) Variant

allele

frequency

Homozygous wild-type, GG Heterozygous, GA Homozygous variant, AA

United States 552 461 90 1 0.083

Canada 67 51 15 1 0.126

Europe 41 29 11 1 0.158

Total (%) 660 (100) 541 (81.9) 116 (17.5) 3 (0.4) 0.092

Table 2 Change of genotyping results from 2015 to 2017 of Papillon dogs

Year Dogs

Tested

PLA2G6 Genotypes (c.1579G > A) Variant

allele

frequency

Homozygous wild-type, GG Heterozygous, GA Homozygous variant, AA

2015 97 69 26 2 0.154

2016 479 400 78 1 0.083

2017 84 72 12 0 0.071

Total (%) 660 (100) 541 (81.9) 116 (17.5) 3 (0.4) 0.092

Raj and Giger Canine Medicine and Genetics (2020) 7:17 Page 3 of 4

It should be noted that once a validated pathogenic

variant is identified in a population, one can safely breed

heterozygotes for the variant (asymptomatic carriers) to

homozygous wildtype dogs (clear dogs, normals). Subse-

quent testing of offspring intended for breeding will then

prevent further production of any affected puppies and

reduce or eliminate spreading of the variant allele in the

breed population. Such strategies also highlight the im-

portance of maintaining a broader gene pool in a small

breed population. Although limited in scope, the ob-

served decline between 2015 and 2019 that likely

occured as a result of screening of the breeding popula-

tion and consequent inform ed breeding practices, shows

the keen interest and willingness of breeders to follow

practices that will avo id production of any puppies af-

fected by NAD.

In conclusion, this survey from North America and

Europe using samples collected between 2015 and 2017,

combined with data obtained from other laboratories be-

tween 2017 and 2019 documents the widespread pres-

ence of the PLA2G6 variant in the Papillon population.

Despite the apparent declining prevalence of the

PLA2G6 variant over the past years, screening of Papil-

lons intended for breeding is recommended unless

cleared by parent testing.

Abbreviations

NAD: Neuroaxonal dystrophy; PLAN: PLA2G6-associated neurodegeneration;

OMIA: Online Mendelian Inheritance in Animals

Acknowledgements

The support and participation of members of the Papillon Club of America

are greatly appreci ated. We also thank Leslie King PhD for scientific editing

of manuscript.

Authors’ contributions

KR did the DNA testing, UG and KR developed the concept, analyzed the

data, and drafted and approved the final manuscript.

Funding

Supported in part by the Papillon Club of America and the NIH grant (OD

010939).

Availability of data and materials

The datasets used and/or analyzed during the current study are available

from the corresponding author on reasonable request.

Ethics approval and consent to participate

Not applicable as only data from samples submitted for clinical diagnostics

were used.

Consent for publication

Not applicable.

Competing interests

The authors are or were associated with the not-for-profit PennGen Labora-

tories, which offer certain DNA and metabolic testing for hereditary diseases

in dogs and cats.

Received: 24 Septemb er 2020 Accepted: 19 November 2020

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