Association of follistatin and cytochrome p450 side-chain cleavage enzyme with polycystic ovarian syndrome

Introduction

Women with polycystic ovarian syndrome (PCOS) produce an abnormal amount of male hormones known as androgens. Among disorders of the reproductive age group, PCOS requires particular attention[1] because 5–10% of the pre-menopausal women are affected by PCOS and its major manifestations are chronic anovulation and hyperandrogenism.[2]

Clinical characteristics and histological features should be analyzed for the symptoms of obesity, bleeding, infertility, as well as miscarriages.[3,4] Biochemically, PCOS can be described with hyperandrogenism and elevated serum luteinizing hormone (LH) as well as hyperinsulinemia. Hyperinsulinemia has been documented as the cause of hyperandrogenism in PCOS women.[5-7] Another study suggested that PCOS increases the risk of cardiovascular as well as diabetic events.[8] The main cause that results in PCOS is still known; however, insulin resistance, inflammation, and genes are associated with excess androgen production in women suffering from PCOS. PCOS can be explained by different pathways. Those pathways include metabolic as well as regulatory avenues and genes should also be assessed for the identification of candidate genes.[9] The familial clustering phenomenon of PCOS cases has been studied for the past 30 years and evidence has suggested that PCOS propagates in the family and the risk for the family members increases up to the extent of 50% but the mode of transmission has not been agreed yet.[10-12,14-16]

The possible candidate genes have been analyzed previously,[17] involved in the pathways of steroid hormones, metabolism and action, gonadotrophic action, obesity and energy regulation, and insulin action.[17-19] Primers were designed to amplify these possibly polymorphic sites, that is, short tandem repeat polymorphism (STRPs) and single nucleotide polymorphism (SNPs), which were located within or adjacent to the candidate genes.[20]

Power estimates can describe the linkage between markers and disease-susceptibility locus as well as the in formativeness of markers. High-density genetic maps have been created which could facilitate positional cloning projects.[21,22] The CYP11A mRNA was observed to be more abundant in the theca cells of individuals with polycystic ovary syndrome as compared to the theca cells of normal individuals. The potential of developing an association is triggered by different factors, and the medical practitioners should evaluate the locus genotypes for the possible penetration of the disease[23] Candidate genes known for PCOS include genes involved in the biosynthesis of androgens, action of insulin, and gonadotrophin[24-28] Genes for different receptors have also been reported as PCOS candidate genes. The key genes associated with dysregulation of androgen production in women include CYP11a, CYP21, CYP17, and CYP19. To study the genetic basis of PCOS, linkage and association studies can be a valid tool.[29] The objective of the present study involves the determination of the Mendelian inheritance of trait, segregation of microsatellite markers for particular loci and determination of the genetic basis of PCOS using linkage analysis and association study strategies.

Materials and Methods

Polymerase chain Reaction (PCR)[20]

Microsatellite markers for the known loci of PCOS as described in Table 1 were obtained from research genetics, Inc. (USA). The cytogenetic information was obtained from genome databases including gdb.org and marshmed.org.

Table 1 37 candidate gene panel for PCOS

The polymerase chain reaction was performed using the following reaction mixture

• Sample DNA 1 μl (microliter)

• 10X PCR buffer 2.5 μl

• MgCl₂ (Magnesium Chloride) 1.5 μl

• Deoxyribonucleotide triphosphate (dNTPs) 0.5 μl

• Forward and reverse primer 0.3 μl

• Taq DNA polymerase 0.3 μl

• PCR water 20.1 μl

A thermo-cycling environment was used for obtaining the mixture as follows:

• 5 min of 90°C for template denaturation

• 40 cycles of amplification each consisting of 3 steps

• 1 min at 90°C for DNA denaturation

• 1 min at 55–57°C for primer annealing

• 1 min at 72°C for extension of complimentary

• DNA strand

• 10 min at 72°C for un-extended strands left

• PCR was performed subsequently.

Results

Pedigree analysis

The six pedigrees proceeded for the present study indicated recessive mode of inheritance. Pedigrees of the strongly associated families, that is, family 2 and family 5 have been described in Figures 1 and 2, respectively. Phenotype of PCOS women in current study exhibit significant hirsutism, obesity acne, acanthosis nigricans and fertility and menstrual issues in 50% moderate hirsutism and acanthosis nigrican in 52.5% women. Family 2 showed strong association with D5S822 at locus (X² = 4.89; P < 0.05) while family 5 depicted strong association with D15S519 (X² = 7.78; P < 0.01). Family 1, 3, 4, and 6 showed weak association with different markers [Tables 2 and 3].

Close

Figure 1

Family 2 Screening

Export to PPT

Close

Figure 2

Screening of family 5

Export to PPT

Table 2 Association of microsatellite markers with families of PCOS

Table 3 Statistical analysis of associated markers

Figures 3 and 4 show the Acrylamide gel photographs of markers for chromosomes in different families to investigate the affected, normal and positioning of each individual. It was observed that 7 individuals that were affected ranged from 4 to 9 and 7 normal individuals ranged from 2 to 6. Two types of marriages were observed in these pedigrees (i) a marriage between PCOS and normal and (ii) between normal and non-PCOS. In both marriages, the data was assembled regarding the inheritance of PCOS and non-PCOS conditions in the offspring. Data revealed a 1:1 Mendelian ratio between normal and non-PCOS marriages [Table 4] while a Mendelian ratio of 3:1 has been observed in marriages between normal and PCOS individuals [Table 5].

Close

Figure 3

Acrylamide gel Photographs of markers for chromosome no.5 with family 2

Export to PPT

Close

Figure 4

Acrylamide gel photographs of markers for chromosome no. 15 family[15]

Export to PPT

Table 4 The goodness of fit into 1:1 Mendelian ratio in marriages between non-PCOS and PCOS individuals

Table 5 The goodness of fit into 3:1 Mendelian ratio in marriages between non-PCOS and PCOS individuals

Association study results

Strong and weak association of different families has been described in Tables 2 and 3.

Discussion

A polycystic ovarian syndrome is considered a complex disorder in a clinical setting. The Present study showed autosomal mode of inheritance in all families. Current study describes that there is a strong association of PCOS locus at chromosome 5 with marker D5S822 (P < 0.05) in family 2 for allele 1 [Tables 1-5]. A study[18,22,23] involved families from European descent (n = 90), Caribbean descent (n = 5), Mexican descent (n = 2), African-American descent (n = 1), and Asian-Indian descent (n=1). The association between PCOS and alleles for four markers (D5S474, D5S822, D5S623, and the SNP in exon 6) was carried out. TDT analysis revealed that 2 markers with the highest X² values were the exon 6 variant (allele 1, X²= 5, P = 0.025) and the D5S623 (allele 11, X² = 4.26, P = 0.039). The expression level of follistatin between PCOS and control subjects was compared but no substantial difference was found. The present study highlighted that PCOS are a complex genetic trait requiring immediate medical attention in Pakistan. Current research indicated a very strong association of PCOS locus with marker D15S519 at chromosome 15 for allele1 (P < 0.01) in the case of family 5 [Table 2]. This marker is derived from the genetic region of CYP11A which has been involved previously in the etiology of PCOS.[18] Their association data indicated that allele variants of CYP11A mediate the development of hyperandrogenemia, which, in turn, is associated with PCOS and hirsutism.[24] Results of family 6 indicated that there is a weak association of PCOS locus at chromosome 19 with marker D19S391 (P =0.05) for allele 1, While a very weak association of PCOS locus at marker D195212 for allele 1 in the case of family 1 has been observed (P > 0.01) [Table 3]. Marker D19S391 has been derived from insulin receptor gene locus while marker D19S212 has been derived from the INSL3 region which represents Leydig insulin-like protein 3. Similar research[31,32] revealed that insulin receptor gene marker D19S884 was significantly associated with PCOS [P = 0.001]. They suggested that the susceptibility gene for PCOS was located on chromosome 19p13.3 in the insulin receptor region and insulin-stimulated androgen secretion from the ovarian stroma. INSR function in the ovary is likely involved in the gender susceptibility of PCOS. Another demonstration revealed[33] that a D19S884 allele shows significant evidence for linkage and association specific to a phenotype of PCOS. A study indicated the involvement of the insulin receptor gene by taking into consideration the tyrosine kinase domain of the insulin receptor gene. They studied 22 patients with PCOS but no abnormalities have been indicated. Molecular scanning of the entire coding region of the gene in 24 hyperinsulinemia subjects was carried out with PCOS but no significant mutation has been detected.[34] They concluded that mutation of the insulin receptor gene is, therefore, unlikely to be a major cause of insulin resistance in PCOS. A similar opinion was given by another study[35] in their study. Present study showed secondary amenorrhea in significant number of women which is indicative of anovulation which depicts problem in their steroid and insulin metabolism. Family 2 2 revealed strong association with D5S822 which is follistatin gene marker. Follistatin is involved in autocrine, paracrine and endocrine control of ovarian folliculogenesis.[29]

Family 5 showed strong association with D15S519 which is involved in steroid hormone control. Candidate genetic loci including CYP11A, CYP17, follistatin, and insulin receptor have been identified by mutation detection, linkage, and case–control association studies. However, traditional linkage analyses have been confounded by extreme phenotypic heterogeneity.[30] In the search of candidate regions for PCOS, many studies were carried out. Follistatin gene was sequenced but subsequent linkage and association studies yielded negative results.[22] A vigorous debate is under consideration about whether PCOS is fundamentally a neuroendocrine or ovarian disorder. Evidence is accumulating in favor of the latter. Thus, it seems that PCOS creates genetic complications and causes abnormalities such as steroidogenesis[25]

It is concluded that different results report the relationship of genes to the risk of PCOS.[1] The mutations concerning PCOS have not been analyzed extensively particularly its effect on the phenotype. Thus, the future researchers must focus on addressing the mutations of PCOS and its impact on phenotype. The complications in the treatment follow an irregular pattern and they require a patient-centered approach and examination.

Conclusion

The current study aimed to determine the association of follistatin and cytochrome p450 side-chain cleavage enzyme (CYP11A) in polycystic ovarian syndrome (PCOS). The study targeted the Pakistani population. A sample of 62 participants based on 6 families was collected to obtain data and analyze it in context with CYP11A and PCOS. Furthermore, the findings showed a strong association of polycystic ovarian syndrome (PCOS) locus at chromosome 5 with marker D5S822 in family 2. The POCS possesses the ability to generate complications in genetics resulting in abnormalities such as steroidogenesis. The study concluded that treatment and diagnosis complications are a major concern in Pakistan and require a more patient-centered approach to treat the disorder and eliminate the complications. Hence, future medical researchers should pay more attention to addressing the mutations of PCOS and its effect on the phenotype. The study also signifies the sensitivity of the relationship between PCOS and the insulin gene marker (D19S884) that is vitally involved in the gender susceptibility in the ovaries. Multiple genes have been examined for the study of PCOS but none of them seem to play a part in PCOS. However, the candidate genes INS VNTR and CYP11A were observed as the encoders of P450 chain cleavage.