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VOLUME 15 NUMBER 1 2022 ISSN 2218-7979 eISSN 2409-370X

International Journal of

Biology

and Chemistry

Al-Farabi Kazakh National University

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EDITORIAL

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International Journal of Biology and Chemistry 15, № 1, 4 (2022)

IRSTI 68.37.31; 34.15.23 https://doi.org/10.26577/ijbch.2022.v15.i1.01

A.A. Malysheva¹ , A.M. Kokhmetova^1* , M.K. Kumarbayeva¹ , D.K. Zhanuzak¹ , A.A. Bolatbekova¹ , Zh.S. Keishilov¹ ,

E.I. Gultyaeva² , A.M. Kokhmetova¹ , V. Tsygankov³ , Y.B. Dutbayev⁴ , S.B. Dubekova⁵

1Institute of Plant Biology and Biotechnology, Almaty, Kazakhstan

2All-Russian Research Institute of Plant Protection, St. Petersburg, Russia

3LLP «Aktobe Agricultural Experimental Station», Aktobe region, Kazakhstan

4Kazakh National Agrarian Research University, Almaty, Kazakhstan

4LLP «Kazakh Research Institute of Agriculture and Plant growing», Almaty, Kazakhstan

*e-mail: [email protected]

(Received 22 April 2022; received in revised form 16 May 2022; accepted 20 May 2022)

Identification of carriers of Puccinia striiformis resistance genes in the population of recombinant inbred wheat lines

Abstract. Stripe rust (yellow rust) caused by Puccinia striiformis (Pst) f. sp. tritici is one of the most dangerous diseases of wheat. Marker assisted selection (MAS) accelerates the selection of resistance gene donors in wheat recombinant inbred lines Almaly/Avocet (S) and to evaluate their response to Pst. Evaluation of seedling resistance to Pst allowed us to select 2 lines simultaneously resistant to races 111Е231 and 7E63.

Race 7E63 was avirulent to most samples, and 111Е231 was highly virulent. All the studied lines demonstrated a resistant and moderately resistant reaction to the causative agent of yellow rust at the adult plant stage (R-MR). Molecular screening revealed the presence of a marker allele associated with the Yr18/Lr34 gene complex in 5 RIL. The frequency of resistant genotypes inheriting the Yr18 gene was 22.7%. The results can be used in MAS wheat breeding programs to increase resistance to yellow rust of wheat.

Key words: wheat, yellow rust, Puccinia striiformis, resistance genes, molecular markers.

Introduction

Wheat (Triticum aestivum L.) is a significant food crop at the global level, and its production is the basis of food security throughout the world [1].

World wheat production currently reaches 777.8 million tons, and world consumption per capita is 67.6 kg/year [2]. On average Kazakhstan produces 18-20 million tons of wheat grain, but output is highly dependent on weather and in recent years has fluctuated between 10 and 17 million tons [3]. Wheat production in Kazakhstan is constrained due to rust diseases (stem, stripe and leaf rus) [4-10], as well as leaf spot diseases (tan spot and rust) [11-16].

Yellow rust caused by Puccinia striiformis Westend f. sp. tritici (Pst) is one of the most dangerous diseases of wheat worldwide. When epiphytotic occur, losses can vary from 20 to 40%

or more [17-18]. A decrease in yield is observed as a

result of a decrease in the number of grains in the ear and grain weight in highly sensitive wheat varieties [4, 19]. In the period from 2009 to 2016, stripe rust epidemics occurred annually in Kazakhstan, and yield losses of susceptible wheat varieties reached 20-50% [4]. The rust pathogen population overcomes the protection of resistant cultivars due to the emergence of virulent pathogen races [18].

The cultivation of resistant varieties is the most economical and environmentally friendly approach, which makes it possible to abandon the use of fungicides and reduce crop losses due to yellow rust [20]. However, traditional breeding methods are not always effective. Marker-assisted selection (MAS) using identified target genes makes the process of developing the wheat cultivars more accurate and reliable [21] MAS methods are effectively used to shorten the breeding cycle, increase resistance to biotic and abiotic stresses and maintain potential

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Int. j. biol. chem. (Online) International Journal of Biology and Chemistry 15, № 1 (2022) A.A. Malysheva et al.

wheat yields [22]. Currently, more than 80 genes for resistance to stripe rust have been identified [22].

Based on recent evaluations in Kazakhstan, genes Yr5, Yr10, Yr15 and Yr18 are still effective [9]. The locus Lr34/Yr18/Pm38 confers partial and durable resistance against the devasting fungal pathogens leaf rust, stripe rust, and powdery mildew. Yr18/

Lr34 genes have been used in breeding programs for a century and so far, no pathogen adaptability has been found [23]. Wheat cultivars containing these genes occupy more than 26 million ha in various developing countries alone and contribute substantially to yield savings in epidemic years [24]. The gene complex Yr18/Lr34 is of great interest as a donor of valuable traits. Therefore, identification of novel sources of resistance in a breeding material is of foremost importance for the effective disease control. In view of these facts, recombinant inbred lines of bread wheat developed in our laboratory were evaluated in this study for yellow rust resistance. The goal of this study was to determine the presence/absence of yellow rust resistance genes in wheat recombinant inbred lines Almaly/Avocet (S) and to evaluate their response to Pst.

Materials and methods

Twenty-two RILs used in this study were established at F₆generation by the single-seed descent method (SSD) from an F₂ family between a high yielding Kazakh winter wheat variety ‘Almaly’

and an Australian spring wheat variety ‘Avocet (S)’. Almaly is a Kazakh variety of common winter wheat, developed in 2002, pedigree [(R6862/50431) xBezostaya1], is widely used as a parent in breeding programs in Kazakhstan and Central Asian countries.

Almaly has a high yield potential and moderately high resistance to 3 species of rust, is a carrier of genes Lr1, Lr28 and Lr34 [6]. The pedigree of the cultivar contains Bezostaya1, a carrier of the leaf rust resistance genes Lr34, Lr3a and Lr13 (https://

maswheat.ucdavis.edu). Second parent is ‘Avocet (S)’, spring wheat cultivar, is considered to be universally susceptible to the three types of rust and is widely used in rust resistance tests. The parents were selected on the basis of their contrasting phenotypic expression of resistance to stripe rust.

The highly susceptible Morocco, as well as the near- isogenic lines (NIL Lr34/TC*6/PI58548 for Yr18) of Thatcher, are used in field and laboratory tests as controls.

It is known that in order to develop a mapping population, it is necessary to cross parents that are contrasting in terms of the target trait. Therefore, the Almaly variety, moderately resistant to the disease, served as the maternal parent, and the Avoset (S) variety, susceptible to stripe rust, was used as the father. This made it possible to obtain a mapping population consisting of 186 RILs with a wide range of genetic diversity for stripe rust, susceptibility to which varied from 0 to 100%. These RILs will be the objects of further studies: on the basis of QTL mapping, genetic loci of quantitative traits associated with resistance to YR of wheat will be identified and mapped. However, in this study, we will study only 22 RILs that demonstrated field resistance to stripe rust. These lines were selected based on their field and laboratory phytopathological screening for resistance to Pst, where cv. Almaty was resistant and Avocet (S) was susceptible.

The phenotyping of the material was carried out in the conditions of the Kazakh Research Institute of Agriculture and Crop Production (KazNIIZiR), Almalybak (43°13’09”N, 76°36’17”E), Almaty region in 2019-2020 cropping season. Each entry was planted in 1 m² plot in the middle of September.

Experiment was conducted using randomized complete block design with two replications and recommended cultural practices were used for trial management. For the replicated data means were calculated. The stripe rust susceptible cultivar Morocco was planted in every 10th row and as a spreader border around the nursery to ensure uniform infection. In mid-April, stripe rust induced susceptible cultivar Morocco, was inoculated with mixed races of Pst at seedling stage in the field in Kazakhstan to serve as spreader of stripe rust pathogen to the experimental plots. Weather conditions in Almaty in 2019 and in 2020 were favorable for the development of stripe rust, and the infection on susceptible checks reached 100S. The resistant assessment was carried out according to the method developed by CIMMYT [25]. Five infection types described as the following:

0 – immune; R – resistant; MR – moderately resistant;

MS – moderately susceptible; and S – susceptible.

Severity of disease was recorded in terms of per cent leaf area infection and pustule type was recorded as response.

Spore collection, storage, and reproduction were then conducted in accordance with the methods of Roelfs et al. [25]. Spores of P. striiformis were used to determine the pathotypes of stripe rust isolated from wheat leaves in the different regions of Kazakhstan.

The determination of race–specific seedling resistance

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6 Identification of carriers of Puccinia striiformis resistance genes in the population of recombinant inbred wheat lines

was performed according to the method of Roelfs et al. (1992) [25] using highly virulent Pst races – 111Е231 (virulence 60%) and 7E63 (virulence 73%). The 111Е231 pathotype was characterized by avirulence to varieties-differentiators with Yr genes SU, SD, Yr10, Yr3v, Yr2, and Sp and virulence to varieties with Yr6, Yr7, Yr1, Yr12, Yr8, 3N, Yr6+, Yr7+, and Yr4+ genes. The 7E63 pathotype showed avirulence to the differentials with SU, SD, Yr10, and Sp genes and virulence to varieties with Yr3v, Yr6, Yr7, Yr1, Yr2, Yr12, Yr8, 3N, Yr6+, Yr7+, and Yr4+

genes. The results were evaluated on 15-20 days according to the Gassner and Straib accounting scale (IT from 0 to 4) [26].

Genomic DNA was extracted at the stage of 3–5-day-old wheat seedlings using the СТАВ method [27]. The presence of the Lr34/Yr18 gene complex was identified using a specific codominant STS marker csLV34 (Yr18) [28], PCR was performed in a Bio-Rad T100TM amplifier (Bio-RAD, Hercules, California, USA). The PCR mixture contained 2.5 µl of genomic DNA (30 ng), 1 µl of each primer (1 pM/

µl) (SigmaAldrich, St. Louis, Michigan, USA), 2.5 µl of dNTP mixture (2.5 mM, dCTP, dGTP, dTTP and dATP (aqueous solution) (Silex CJSC, Russia), 2.5 µl MgCl2 (25 mM), 0.2 µl Taq polymerase (5 units.

mcl) (CJSC “Silex”, Russia), 2.5 mcl 10X buffer for PCR and 12.8 mcl ddH20. PCR was performed at initial denaturation of 94 °C for 5 minutes, 40 cycles:

94°C – 40 s., 55°C – 30 s., 72°C – 1 min., final elongation at 72 °C – 7 min. The separation of PCR products was carried out in a 2% agarose gel using a TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8) with the addition of ethidium bromide. The lengths of the amplicon fragments were determined using a 100-bp DNA marker. Visualization of the results was performed using the gel documentation system (Gel Doc XR+, BIO-RAD, Hercules, California, USA)

Results and discussion

The parent variety Almaly is characterized by high yields and a moderate degree of resistance to stripe rust. There is a variety of Bezostaya 1 in Almaly’s pedigree, which is a carrier of the Yr18 gene [29]. The Australian variety Avocet (S), on the contrary, has a high degree of susceptibility to stripe rust. The results of phytopathological evaluation of the breeding material under artificial infectious background allowed us to select 22 RIL Almaly/Avocet(S) lines that showed a high degree of resistance to the stripe rust pathogen Pst at the adult plant stage (0-20MR). From the two parents, ‘Almaly’ showed a moderate degree of resistance (20MR), while ‘Avocet(S)’ and the susceptible control Morocco showed a high degree of susceptibility (100S, both) (Table 1).

The other control line NIL Lr34/TC*6/PI58548 for Yr18, the carrier of gene Yr18 showed a moderate susceptibility with low severity (10MS).

In greenhouse experiment for seedling resistance lines investigated showed a different infectious type (ITs) for the two races of Pst. Avirulence of race 7E63 was shown to the majority of the studied RIL entries (91%) (IT-0), while 2 lines (9%) had only moderate resistance to the pathogen (IT-2). Evaluations for race 111Е231 showed the following results: 14 RILs had a high degree of susceptibility (IT-4), 6 lines were moderately susceptible (IT-3), one line was moderately resistant and one immune (IT-2 and IT-0, respectively). The susceptible parent ‘Avocet (S)’ and the control ‘Morocco’ both showed a high susceptibility (IT-4) for both races, while the parent

‘Almaty’ and the isogenic line Lr34/TC*6/PI58548 (carrier of gene Yr18) were moderately resistant (IT- 2) to race 111Е231 and was immune (IT-0) to race 7E63.

Table 1 – Yellow rust severity and the presence of the Yr18 gene in the genotypes of the population of recombinant inbred lines Almaly/

Avocet

# Genotype Origin^a APR^b 111Е231^c 7E63^c CSLV34^d Yr gene detected based

on linked marker

♀ Almaly KZ 20MR 2 0 + Yr18

♂ Avocet (S) AUS 100S 4 4 - -

1 RIL Al/Av(S)-951-664 KZ 0 3 0 - -

2 RIL Al/Av(S)-1094-817 KZ 0 3 0 + Yr18

3 RIL Al/Av(S)-1085-808 KZ 20MR 3 0 - -

4 RIL Al/Av(S)-1086-809 KZ 10MR 3 0 - -

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# Genotype Origin^a APR^b 111Е231^c 7E63^c CSLV34^d Yr gene detected based

on linked marker

5 RIL Al/Av(S)-852-560 KZ 20R 4 0 + Yr18

6 RIL Al/Av(S)-882-589 KZ 0 4 0 - -

7 RIL Al/Av(S)-883-590 KZ 0 4 0 - -

8 RIL Al/Av(S)-968-682 KZ 20MR 4 0 - -

9 RIL Al/Av(S)-975-691 KZ 0 4 0 - -

10 RIL Al/Av(S)-976-692 KZ 0 4 0 - -

11 RIL Al/Av(S)-982-698 KZ 20MR 4 0 - -

12 RIL Al/Av(S)-986-702 KZ 0 4 0 - -

13 RIL Al/Av(S)-993-710 KZ 20MR 4 0 - -

14 RIL Al/Av(S)-994-711 KZ 20MR 4 0 - -

15 RIL Al/Av(S)-1058-781 KZ 5R 4 0 - -

16 RIL Al/Av(S)-1097-821 KZ 0 4 0 - -

17 RIL Al/Av(S)-1099-823 KZ 0 4 0 - -

18 RIL Al/Av(S)-1053-776 KZ 10MR 3 2 + Yr18

19 RIL Al/Av(S)-1054-777 KZ 10MR 3 2 + Yr18

20 RIL Al/Av(S)-1052-775 KZ 0 4 2 + Yr18

21 RIL Al/Av(S)-1067-790 KZ 0 0 0 - -

22 RIL Al/Av(S)-1009-725 KZ 20MR 2 0 - -

Controls

23 NIL Lr34/TC*6/PI58548 USA 10MS 2 0 + Yr18

24 Morocco MА 100S 4 4 - -

a – Origin include countries and organizations: KZ – Kazakhstan, AUS – Australia, USA – United States of America, MА – Marocco, Almaty – Institute of Plant Biology and Biotechnology;

b – Values indicate severity;

c – IT– infection typeaction type;

d – “+”, “-” – indicate the presence and absence allele of corresponding gene, respectively.

Continuation of the table

The STS marker csLV34 is a diagnostic marker for the Yr18/Lr34 gene complex, which allows determining the allelic state of a gene. Two alleles demonstrate amplicons that are clearly distinguishable in the agarose gel (150 bp for the dominant state and 229 bp for the recessive state). Molecular screening revealed the presence of a marker allele associated with Yr18/Lr34 in 5 RIL Almaly/Avocet(S) (1094- 817, 852-560, 1053-776, 1054-777, 1052-775) (Table 1, Figures 1 and 2). Thus, the frequency of resistant genotypes inheriting the Yr18 gene was 22.7%. The heterozygous state of alleles of the Yr18/Lr34 gene was not detected. (Figures 1 and 2.)

The complex of resistance genes Yr18/Lr34/ Sr57/

Pm38 is important in the breeding of resistant varieties to

yellow, leaf, and also partially to stem rust and powdery mildew [30]. This gene retains its effectiveness for about 100 years, which is due to the molecular characteristics of the defense mechanism. The activity of Yr18/Lr34 induces necrosis of the tip of the flag leaf.

However, the observed reaction cannot be used as a phenotypic marker for Yr18/Lr34, which also manifests itself with other resistance genes (such as Lr46/Yr29/Pm39) [31], which indicates the need for molecular screening. Yr18/Lr34 is a gene for adult plant resistance (APR), nevertheless able to impart resistance at the seedling stage to some rust races [32]. It is possible that the processes induced by Yr18/

Lr34 make the tissue less favorable for biotrophic pathogens [33].

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Figure 1 – Wheat DNA amplification products using primers to the STS csLV34 locus linked to the Yr18/Lr34 resistance gene.

Note: 1 – Almaly, 2 – Avocet(S), 3 –14 RIL Almaly/Avocet: 3 – RIL 951-664, 4 – RIL 1094-817, 5 – RIL 1085-808, 6 – RIL 1086-809, 7 – RIL 852-560, 8 – RIL 882-589, 9 – RIL 883-590, 10 – RIL 968-682, 11 – RIL 975-691,

12 – RIL 976-692, 13 – RIL 982-698, 14 – RIL 986-702; 15 – Yr18/NIL-Lr34/TC-6/PI58548, 16 – dd H₂O, M – Gene –Ruler 100 bp DNA Ladder

Figure 2 – Wheat DNA amplification products using primers to the STS csLV34 locus linked to the Yr18/Lr34 resistance gene (continued). Note: 1 – Almaly, 2 – Avocet(S), 3 –14 RIL Almaly/Avocet:

3 – RIL 993-710, 4 – RIL 994-711, 5 – RIL 1058-781, 6 – RIL 1097-821, 7 – RIL 1099-823, 8 – RIL 1053-776, 9 – RIL 1054-777, 10 – RIL 1052-777, 11 – RIL 1067-790, 12 – RIL 1009-725; 13 – Yr18/NIL-Lr34/TC-6/PI58548,

14 – dd H₂O, M – Gene –Ruler 100 bp DNA Ladder

It was previously shown that Yr18/Lr34 provides a sufficiently high level of resistance in the conditions of an artificial epidemic. The effect of this gene is enhanced in combination with other resistance genes, such as Yr5 and Yr10 [5]. Molecular screening revealed 5 (22.7%) carriers of the Yr18 gene (RIL Almaly/Avocet(S) 1094-817, 852-560, 1053-756, 1054-777 and 1052-775) from 22 studied samples, which indicates a high degree of its inheritance among resistant lines. No resistance genes were found in 71% of the studied samples, however, a high level of immunity to the pathogen Pst was noted in the field (0-20MR).

Conclusion

The results of field phytopathological screening of collection of RILs Almaly/Anza in field conditions to yellow rust caused by Pst allowed to select 22 resistance wheat lines, presumably carriers of resistance genes. The results of the evaluation of the selected lines of seedling resistance showed a contrasting reaction to two races of yellow rust: race 7E63 was avirulent to most samples, and 111Е231 was highly virulent. Evaluation of the resistance

of seedlings to Pst allowed us to identify 2 lines that were simultaneously resistant to both races.

Molecular screening revealed the presence of a marker allele associated with the Yr18/Lr34 gene complex in 5 samples. The frequency of resistant genotypes inheriting the Yr18 gene was 22.7%.

The data obtained indicate the possibility of increasing the resistance of the material due to hybridization with productive wheat varieties and lines. The selected lines can be used in breeding programs to breed varieties resistant to yellow rust.

Marker selection methods significantly simplify the process of selecting donors of resistance genes, which has a positive effect on the prospects for the development of the agricultural sector in Kazakhstan.

Acknowledgments

The research was carried out within the framework of the OR11465424 program “Development and implementation of highly effective diagnostic systems for identifying the most dangerous diseases and increasing the genetic potential of crop resistance”, task 03 “Development of DNA markers for creating

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a system for identifying resistance genes based on mapping loci of quantitative traits associated with resistance to yellow rust in the population of recombinant inbred wheat lines” for 2021-2022 with the financial support of the Ministry of Science and Higher Education of the Republic of Kazakhstan.

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IRSTI 34.23.33 https://doi.org/10.26577/ijbch.2022.v15.i1.02

A. Perfilyeva¹ , K. Bespalova^1,2* , G. Abylkassymova¹ , B. Bekmanov^1,2 , L. Djansugurova^1,2

1Institute of Genetics and Physiology, Almaty, Kazakhstan

2Al-Farabi Kazakh National University, Almaty, Kazakhstan

*e-mail: [email protected]

(Received 12 May 2022; received in revised form 24 May 2022; accepted 31 May 2022)

RS2710102 polymorphism of the CNTNAP2 gene is related to autism susceptibility in a Kazakhstani population

Abstract. Autism spectrum disorders (ASDs) represent serious mental development disorders characterized by deficits in verbal and non-verbal communication, reciprocal social interactions and stereotypical behaviors. Genetically determined pathologies of neurodevelopment and synaptic functioning are increasingly considered to be a cause of ASDs. Contactin associated protein-like 2 (CNTNAP2) gene encodes a protein, which plays an essential role in brain development. Genetic variations in the CNTNAP2 gene can perturb its functions, contributing to the genetic predisposition to ASDs. The study aimed to investigate an association of the CNTNAP2 rs2710102 with ASDs in a Kazakhstani population. The study involved patients diagnosed with ASDs and healthy controls of Kazakhstani origin. PCR-RFLP assay was used for the genotyping rs2710102 CNTNAP2 SNP. The distribution of the rs2710102 genotype was under the Hardy-Weinberg equilibrium in both cases and controls. C allele and СС genotype were associated with a significantly increased risk of ASDs (OR = 3.04, 95% CI = 1.96-4.72, p<0.001 and OR = 6.41, 95% CI = 2.47-16.63, p<0.001, respectively), which was also confirmed for males (OR = 2.25, 95% CI = 1.23-4.10, p=0.007 and OR = 2.95, 95% CI = 1.06-8.18, p=0.029, respectively) and females (OR = 4.75, 95% CI

= 1.91-11.77, p<0.001 and OR = 7.20, 95% CI = 0.89-58.53, p=0.002, respectively). In contrast, there was no statistically significant association of the rs2710102 with deficits of verbal communication in ASD patients. The obtained results provide the first significant link between rs2710102 CNTNAP2 and autism susceptibility in Asian populations.

Key words: Autism spectrum disorders (ASDs), CNTNAP2 gene, single-nucleotide polymorphism, genetic susceptibility, Kazakhstani population, neurodevelopment.

Introduction

Autism spectrum disorders (ASDs) are a group of heterogeneous neurodevelopmental diseases, which are characterized by deficits in verbal and non-verbal communication, reciprocal social interactions and stereotypical behavior. In Kazakhstan, as everywhere in the world, the number of children with ASDs has been increasing annually. In Kazakhstan, according to the Service of psychological, medical and pedagogical consultation, autism was diagnosed in 3820 children (in the 2020 year), however, according to the international experts, the real number of children with ASD significantly exceeds this figure.

For, according to the Institute of Autism at Oregon State University (USA), in our country 59 thousand children have autism spectrum disorders.

Genetic factors are one of the main components of ASDs. Literature shows hundreds of candidate genes

for ASDs, which may be a subject of closer attention and deserve a separate study. Here, we focused on the contactin associated protein-like 2, which has been proposed to be a candidate gene for ASDs [1].

CNTNAP2 gene is located in 7q35–36 and encodes the transmembrane protein Caspr2, which is a member of the neurexin family. This protein is localized at the juxtaparanodes of myelinated axons providing interactions between neurons and glia during nervous system development. It is also involved in clustering K+ channels in myelinated axons [2].

Several genetic, neurobiological and mouse model studies have supported the role of CNTNAP2 in ASDs and other related neurodevelopmental disorders. In the Old Order Amish community, the mutations of CNTNAP2 have been identified as causes of symptomatic childhood-onset epilepsy, characterized by the presence of neuronal migration

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12 RS2710102 polymorphism of the CNTNAP2 gene is related to autism susceptibility in a Kazakhstani population

abnormalities, intellectual disability, language regression, seizures, hyperactivity, impulsive/

aggressive behavior, as well as ASDs [3].

In mouse models, CNTNAP2 deficiency has shown extraordinary similarity to the main deficits of behavioral and cognitive functions of ASD patients.

Neurophysiological features, such as cortical neuronal migration abnormalities, observed in CNTNAP2-/- mice, further supported the involvement of the CNTNAP2 gene in ASD pathologies [4]. The migration of neurons in the developing cerebral cortex plays a key role in the development of brain and brain networks [5]. A number of neuroimaging studies demonstrated abnormal connectivity and altered brain networks in individuals with ASDs [6–8].

Moreover, mouse model studies have demonstrated a capacity of anti-Caspr2 auto-ABS to generate ASD- like behavior [9, 10].

Besides, the CNTNAP2 gene is expressed in language-related cortical areas [11]. Hence, given the speech impairment in autism, CNTNAP2 becomes a strong candidate gene for autism.

Genetic variations in the CNTNAP2 gene can affect its functions contributing to the genetic predisposition to ASDs. It has been shown that the loss of even one CNTNAP2 allele leads to elicit axonal growth alterations [12]. Several common and rare variants of CNTNAP2 have been reported to be associated with autism as well as related to phenotypes such as intellectual deficiency, impaired language, abnormal social behavior, epilepsy and schizophrenia [13–17].

The single-nucleotide polymorphism (SNP) rs2710102 is located at intron 13. There are still insufficient data on the relation of this common variant with ASDs in Asian populations. Therefore, in the present study, we carried out a case–control association study between rs2710102 and ASDs in a Kazakhstani population.

Materials and methods

The study involved patients diagnosed with ASDs and healthy controls of Kazakhstani origin. A total of 51 healthy individuals and 280 ASD patients had been recruited in rehabilitation centers of Kazakhstan starting from March 2018 to December 2020.

Collection of the clinical material in families with children with ASD was carried out in the “Autism Pobedim” Foundation based on the Memorandum of Cooperation, as well as in public funds, working with ASD children in Almaty, Astana, Zhezkazgan, Karaganda, Kokshetau, Kyzyl-Orda, Pavlodar, Petropavlovsk, Temirtau, Ust-Kamenogorsk, Shymkent, Ekibastuz and South Kazakhstan region.

Buccal epithelium samples were collected from patients with ASDs, as well as from healthy individuals using sterile cotton-tipped applicators.

The collected material was transported to the Institute of General Genetics and Cytology in a portable refrigerated container and frozen at -80° C for further molecular-genetic studies.

The collection of biomaterial was conducted exclusively voluntarily after signing an informed consent by at least one of the parents. The protocol of the study was approved by the Ethics Committee of S. Asfendiyarov Kazakh National Medical University (Protocol #57 from 05.09.2017).

The clinical diagnosis of autism was established by senior psychiatrists and assessed by the CARS for children over 3 years and the M-Chat-R for children under three years old. CARS and M-Chat-R have been used as standardized, investigator-based instruments for the detection of ASDs [19, 20]. ASD patients with M-CHAT or CARS scores in the low- risk range were excluded from the study. Additional exclusion criteria were a recognizable neurological or genetic disorder (Rett syndrome, Fragile X syndrome and others), noncitizens of Kazakhstan.

For the control group, the exclusion criteria were the presence of autism or other mental disorders in personal and family history, M-CHAT or CARS scores in the medium/high-risk range, noncitizens of Kazakhstan.

Verbal communication in the ASD group was assessed by interviewing parents of ASD children and classified as without (verbal communication is appropriate for age and situation) or with speech disorders (delayed speech, echolalia, meaningless speech, etc.).

DNA from buccal swabs was isolated using a DNA extraction kit (AmpliSens). DNA samples were stored at -20°C and -80°C. PCR-RFLP assay was used for the genotyping rs2710102 CNTNAP2 SNP as described earlier [18].

Statistical analysis was performed using

“Case-Control Study Estimating Calculator” by TAPOTILI company (Laboratory of Molecular Diagnostics and Genomic Dactyloscopy of

“GosNIIGenetika” State Scientific Centre of Russian Federation). Hardy-Weinberg equilibrium (HWE) test was used to compare the observed and expected genotype frequencies. Relative risks were estimated by odds ratios (OR) with a logistic regression 95% confidence interval. Statistical analysis considering the models of inheritance–

the multiplicative, dominant, and recessive – was conducted for the examined SNP. P < 0.05 was considered statistically significant.

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Results and discussion

General Сharacteristics of Patients.

Characteristics of the ASD patients and healthy controls are summarized in Table 1. The ethnic heterogeneity of both groups was Kazakh, Russian and other Europeans and Asians. The mean age for

the ASD group and control subjects was 7.41±8.39 years (range 2-34 years) and 7.08±4.42 years (range 1-39 years), respectively. In the ASD group, 79.3%

were males and 20.7% were females. In the control group, 47.1% were males and 52.9% were females.

173 ASD patients had speech disorders and 68 without.

Table 1 – Characteristics of ASD and control groups

Characteristic ASD N (%) Controls N (%)

Sample size 280 51

Ethnicity

Kazakh 180 64.3 39 76.5

Russian 68 24.3 5 9.8

Other Europeans 3.2 9.0 4 7.8

Other Asians 8.2 23.0 3 5.9

Age (years) Median 7.41±8.39 7.08±4.42

Gender Male 222 79.3 24 47.1

Female 58 20.7 27 52.9

Analysis of the Association of the rs2710102 CNTNAP2 Polymorphism with the Risk of ASD in a Kazakhstani Population. The genotype distributions of the rs2710102 polymorphism were under Hardy- Weinberg equilibrium (HWE) for both control (p=0.585) and ASD cases (p=0.818). The distribution of rs2710102 CNTNAP2 genotypes is presented in Table 2.

As shown in Table 2, the CNTNAP2 C allele and СС genotype were associated with a significantly increased risk of ASD (OR = 3.04, 95% CI = 1.96- 4.72, p<0.001 and OR = 6.41, 95% CI = 2.47-16.63, p<0.001, respectively). Furthermore, a significantly increased risk of ASD was found for CC+CT genotypes versus the TT genotype in the dominant model (OR = 3.56, 95% CI = 1.84-6.89, p<0.001) and for CC genotype versus the combined variant

of CT+TT genotypes in the recessive model (OR = 6.41, 95% CI = 2.47-16.63, p<0.001).

Due to a gender difference in rates of ASDs we further calculated the association of the rs2710102 CNTNAP2 polymorphism with ASD risk in the sub- groups stratified into genders (Table 2). C allele and СС genotype showed a significant association with ASD in both males (OR = 2.25, 95% CI = 1.23- 4.10, p=0.007 and OR = 2.95, 95% CI = 1.06-8.18, p=0.029, respectively) and females (OR = 4.75, 95%

CI = 1.91-11.77, p<0.001 and OR = 7.20, 95% CI = 0.89-58.53, p=0.002, respectively). Furthermore, the C allelotype (CC+CT genotypes) had a high risk for ASD development in both male and female patients in the dominant model (OR = 2.85, 95% CI = 1.09- 7.49, p=0.027 and OR = 7.03, 95% CI = 2.16-22.88, p=0.001, respectively).

Table 2 – Genotype and allele distributions of CNTNAP2 in ASD patients and controls rs2710102

CNTNAP2 ASD Patients

280 Controls

51 OR 95% CI p-Value

СС 115 5 6.41 2.47-16.63

<0.001

СТ 125 27 0.72 0.39-1.30

ТТ 40 19 0.28 0.15-0.54

Dominant model

СС+СТ 240 32 3.56 1.84-6.89

<0.001

ТТ 40 19 0.28 0.15-0.54

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rs2710102

CNTNAP2 ASD Patients

280 Controls

51 OR 95% CI p-Value

Recessive model

СС 115 5 6.41 2.47-16.63

<0.001

СТ+ТТ 165 46 0.16 0.06-0.40

Allele

C 355 37 3.04 1.96-4.72

<0.001

T 205 65 0.33 0.21-0.51

Male

СС 97 5 2.95 1.06-8.18

0.029

СТ 97 12 0.78 0.33-1.80

ТТ 28 7 0.35 0.13-0.92

Dominant model

СС+СТ 194 17 2.85 1.09-7.49

0.027

ТТ 28 7 0.35 0.13-0.92

Recessive model

СС 97 5 2.95 1.06-8.18

0.031

СТ+ТТ 125 19 0.34 0.12-0.94

Allele

C 291 22 2.25 1.23-4.10

0.007

T 153 26 0.44 0.24-0.81

Female

СС 18 1 7.20 0.89-58.53

0.002

СТ 28 5 2.24 0.70-7.17

ТТ 12 11 0.14 0.04-0.46

Dominant model

СС+СТ 46 5 7.03 2.16-22.88

0.001

ТТ 12 12 0.14 0.04-0.46

Recessive model

СС 18 0 7.20 0.89-58.53

0.036

СТ+ТТ 40 17 0.14 0.02-1.13

Allele

C 64 7 4.75 1.91-11.77

<0.001

T 52 27 0.21 0.08-0.52

OR analysis was performed to evaluate the effect of rs2710102 CNTNAP2 on verbal communication in ASD subjects. As shown in Table 3, there was no statistically significant association of the polymorphism with speech impairments in patients with ASD in the general group, as well as in males or females.

We carried out a case-control association study of the rs2710102 CNTNAP2 in 280 patients and 51 controls to assess the genetic contribution of this

genetic variant to ASDs in a Kazakhstani population.

The results of the study showed significant associations between rs2710102 CNTNAP2 and autism both in males and females. Further analysis found no significant association between the rs2710102 CNTNAP2 and speech impediments in ASD patients.

Discordant results have been reported by previous studies. No association was reported between the rs2710102 CNTNAP2 and autism in

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640 trios of Han Chinese descent [19]. No significant differences between the frequencies of the CC risk genotype were demonstrated in the 210 autistic patients and 200 controls representing a Brazilian population [18]. A study of 67 autism cases and 100 controls did not find significant associations between the rs2710102 CNTNAP2 gene polymorphism and autism in an Iranian population [20]. A case–

control association study of 322 Spanish autistic patients and 524 controls found no association of this polymorphism with autism [21]. Finally, the rs2710102 variant was not significantly associated with autistic-like traits in a Swedish study of 12,319 subjects [22]. Moreover, an updated meta-analysis found no association between the rs2710102 CNTNAP2 and autism [19].

Table 3 – The relation between rs2710102 CNTNAP2 and verbal communication in ASD subjects

rs2710102 CNTNAP2

ASD Patients with

speech disorders ASD Patients without

speech disorders OR 95% CI p-Value

173 68

СС 77 25 1.38 0.77-2.46

0.298

СТ 70 35 0.64 0.36-1.13

ТТ 26 8 1.33 0.57-3.10

Dominant model

СС+СТ 147 60 0.75 0.32-1.76

0.512

ТТ 26 8 1.33 0.57-3.10

Recessive model

СС 77 25 1.38 0.77-2.46

0.273

СТ+ТТ 96 43 0.72 0.41-1.29

Allele

C 224 85 1.10 0.73-1.66

0.645

T 122 51 0.91 0.60-1.37

Male

СС 65 21 1.66 0.88-3.13

0.115

СТ 50 31 0.52 0.28-0.97

ТТ 19 6 1.43 0.54-3.79

Dominant model

СС+СТ 115 52 0.70 0.26-1.85

0.469

ТТ 19 6 1.43 0.54-3.79

Recessive model

СС 65 21 1.66 0.88-3.13

0.116

СТ+ТТ 69 37 0.60 0.32-1.14

Allele

C 180 73 1.20 0.76-1.90

0.422

T 88 43 0.83 0.53-1.31

Female

СС 12 4 0.67 0.16-2.80

0.805

СТ 20 4 1.58 0.38-6.48

ТТ 7 2 0.88 0.15-5.05

Dominant model

СС+СТ 32 8 1.14 0.20-6.59

0.881

ТТ 7 2 0.88 0.15-5.05

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rs2710102 CNTNAP2

ASD Patients with

speech disorders ASD Patients without

speech disorders OR 95% CI p-Value

173 68

Recessive model

СС 12 4 0.67 0.16-2.80

0.579

СТ+ТТ 27 6 1.50 0.36-6.31

Allele

C 44 12 0.86 0.32-2.35

0.772

T 34 8 1.16 0.43-3.15

Contrary, some studies suggested associations between the rs2710102 SNP and predisposition to ASDs. A relationship between frontal lobar connectivity and rs2710102 genetic variant was demonstrated by functional neuroimaging, which indirectly confirms its contribution to ASDs [23].

A positive association of the rs2710102 with ASDs has been found in a study of 152 families from the Autism Genetic Resource Exchange [24].

Besides, previous studies have demonstrated the relation of the rs2710102 with language problems, signifying its essential role in ASD pathogenicity.

This SNP was associated with non-word repetition [25] and specific language impairment [26]. The risk C allele of rs2710102 was significantly associated with a delayed onset of speech, as measured by the

“age at the first word”, in ASD children [27]. Non- autistic homozygous for the C allele demonstrated significantly increased activation in contralateral areas of traditional left-sided language regions: the frontal operculum and middle temporal gyrus [28].

Finally, it was suggested that rs2710102, as a part of specific 4-SNP haplotypes, may influence early language development in the general population [29].

In contrast, two studies failed to replicate positive results on the association between the rs2710102 and impaired language development in ASD patients [21, 22]. Similarly, we also found no association between CNTNAP2 and speech disorders. The reason for this could be insufficient tools to assess speech impairment based on only interviewing parents.

Nevertheless, our results indicate a significant statistical association between the rs2710102 variant and autism in the general group and for both men and women. It is well known that genetic heterogeneity of different populations affects the results of case- control association studies. In this way, the analysis

of genetic predisposition to ASD may require different genetic markers for different populations.

The majority of case-control studies of the rs2710102 variant and autism association were performed in European and American populations. To the best of our knowledge, only two studies have been carried out in Asian populations (Chinese and Iranian) so far [19, 20]. This circumstance can explain the discrepancy between our data and the data of the above-mentioned studies.

In addition, the limitations of this study must be taken into account. The control sample is small compared to the ASD sample and not matched for gender with the ASD sample (males in ASD is 79,3% and controls is 47,1%). Subdivision of all the individuals into male and female groups, as well as into groups with and without speech disorders resulted in relatively small sample sizes, so the power of these subgroup results was < 80%, indicating that additional high-level studies are still needed. The current work investigated only one polymorphism of the CNTNAP2 gene. However, we cannot exclude a possibility that other variants in the CNTNAP2 gene may be involved in ASDs and language impairment.

Further population-based studies that will investigate the effect of genetic variations on ASDs are needed to better understand the genetics of autism and related disorders.

Conclusion

Genetically determined pathologies of neurodevelopment and synaptic functioning are increasingly considered to be a cause of ASDs.

Contactin associated protein-like 2 (CNTNAP2) gene encodes a protein, which plays an essential role in brain development. Genetic variations in the CNTNAP2 gene can perturb its functions, contributing to the genetic predisposition to ASDs.

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In the current study, we provide the first significant link between rs2710102 CNTNAP2 and autism susceptibility in Asian populations. Our results suggest that the SNP rs2710102 of the CNTNAP2 gene may be associated with autism susceptibility in Kazakhstani population, but it not seems to be involved with speech disorders in the same population.

Acknowledgments

This work was supported by the grant OR11465435 received from the Committee of Science, Ministry of Education and Science of Republic of Kazakhstan in 2021. The authors are grateful to the parents and children participating in the study, as well as the heads of the rehabilitation centers, who, through recruitment, have made a valuable contribution to the success of the study. They would particularly like to thank Demeuova Regina, Darmenova Ainur, Seitova Adina, Kvan Olga, Zhumashev Gamali, as well as Perfilyeva Yuliya for critical reading of the manuscript.

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