A primate-specific (GCC) repeat in the untranslated region of SMAD9 undergoes natural selection and links with late-onset neurocognitive disorder in human

doi:10.21203/rs.3.rs-1565676/v1

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A primate-specific (GCC) repeat in the untranslated region of SMAD9 undergoes natural selection and links with late-onset neurocognitive disorder in human

https://doi.org/10.21203/rs.3.rs-1565676/v1

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Across numerous primate species and tissues, SMAD9 (SMAD Family Member 9) gene, reaches the highest level of expression in the human brain. This gene contains a short tandem repeat (STR) of (GCC) motifs at the interval between +1 and +60 of the transcription start site (ENST00000379826.5 SMAD9-202), which is in the 1st percent of high-ranking (GCC)-repeats in respect of length. Here we sequenced this (GCC)-repeat in a sample of 334 Iranian individuals, consisting of late-onset neurocognitive disorder (NCD) (N=167) and controls (N=167). We detected two predominant alleles of 7 and 9 repeats, with allele frequencies ranging from 0.42 and 0.54, and three other alleles of frequencies <0.03 across the human samples studied. The frequency ratio of the (GCC)7 and (GCC)9 alleles was in the reverse order in the NCD group vs. controls, resulting from the excess of (GCC)7 in the former (p=0.009). Five genotypes, predominantly consisting of the (GCC)7 and lacking (GCC)9 were detected in the NCD group only (Mid-p=0.007), and four genotypes consisting of (GCC)9 and lacking (GCC)7 were detected in the control group only (Mid-p=0.003). Those genotypes encompassed 4% of the genotype pool across the human samples studied. The (GCC)-repeat specifically expanded in primates, and reached maximum length in human. In conclusion, we report a novel potential locus for late-onset NCD at the human 5′ UTR (GCC)-repeat. Primate-specificity of this STR, predominant abundance of two alleles across the human samples studied, skewing of those alleles in the NCD group, and enrichment of genotypes consisting of the risk and protective alleles that were specific to the NCD and control groups, respectively, indicate that alleles at this locus have undergone natural selection. Reported instances of STR allelic natural selection at non-coding loci in human are rare, and the SMAD9 (GCC)-repeat provides a potentially valuable locus to further test this phenomenon.

SMAD9

(GCC)-repeat

short tandem repeat

natural selection

late-onset

neurocognitive disorder

An increasing wealth of research supports vast implications of short tandem repeats (STRs) (also known as microsatellites/simple sequence repeats) in evolutionary, biological, and pathological terms ^1–10. However, STRs remain underappreciated in comparison to single nucleotide substitutions ^11,12, partly because of their repetitive nature and hardship of accurate allele calling with the currently available methods.

Across various categories of STRs, (CGG)/(GCC) repeats are overrepresented in the exonic regions of the human genome, and are mainly attended because of their involvement in neurological disorders ^13–16. The human SMAD9 (SMAD Family Member 9) (also known as PPH2, MADH6, MADH9, SMAD8, SMAD 8A, SMAD8B, and SMAD8/9SMAD8), maps on chromosome 13, at 13q12-q14, and contains an annotated (GCC)9 in the interval between + 1 to + 60 of the transcription start site (TSS) (ENST00000379826.5 SMAD9-202), which is in the top 1 percent of (GCC)-repeats in respect of length ¹⁷. Across all annotated primate species and tissues, SMAD9 reaches maximum expression in the human brain (https://www.ncbi.nlm.nih.gov/IEB/Research/Acembly) ¹⁸. Among a number of critical genes, SMAD9 is in the top 5 genes, silencing of which inhibits angiogenic transformation as well as cell migration of human brain micro endothelial cells ¹⁹. The angiogenic potential declines in the process of aging, and stimulation of angiogenesis can improve late-onset cognitive disorders (NCDs) such as Alzheimer’s disease (AD)²⁰.

Here we sequenced the SMAD9 (GCC)-repeat in a sample of humans, consisting of late-onset NCDs and controls. We also studied the status of this STR across vertebrates.

Subjects

Three hundred thirty-four unrelated Iranian subjects of ≥60 years of age, consisting of late-onset NCD patients (DSM-5) ²¹ (N=167) and controls (N=167) were recruited from the provinces of Tehran, Qazvin, and Rasht. In each NCD case, the Abbreviated Mental Test Score (AMTS) ^22,23 was implemented; AMTS<7 was an inclusion criterion for NCD, medical records were reviewed in all participants, and brain CT-scans were taken where possible. Furthermore, in a number of subjects, the Mini-Mental State Exam (MMSE) Test ²³ was implemented in addition to the AMTS. A score of <24 was an inclusion criterion for NCD. The Persian version of the AMTS is a valid cognitive assessment tool for older Iranian adults, and can be used for NCD screening in Iran ²⁴. The MMSE used is also sufficiently accurate to detect patients with cognitive impairment, particularly those with dementia ²⁵. The control group was selected based on cognitive AMTS of >7 and MMSE>24, lack of major medical history, and normal brain CT-scan where possible. The cases and controls were matched based on age, gender, and residential district. The subjects' informed consent was obtained (from their guardians where necessary) and their identities remained confidential throughout the study. The research was approved by the Ethics Committee of the Social Welfare and Rehabilitation Sciences, Tehran, Iran, and was consistent with the principles outlined in an internationally recognized standard for the ethical conduct of human research. All methods were performed in accordance with the relevant guidelines and regulations.

Allele and genotype analysis of the SMAD9 (GCC)-repeat.

Genomic DNA was obtained from peripheral blood using a standard salting out method. PCR reactions for the amplification of the human SMAD9 (GCC)-repeat were set up with the following primers: Forward: GCTGCGAGGAGTACTACCC

Reverse: CACGTCTTACCTGTCCCCG

PCR reactions were carried out in a final volume of 20 µl, at a final concentration of 30% high-GC buffer, in a thermocycler (Peqlab-PEQStar) under the following conditions: initial denaturation at 95 ◦C for 5 min, 40 cycles of denaturation at 95 ◦C for 45 s, annealing at 55 ◦C for 45 s, and extension at 72 ◦C for 1 min, and a final extension at 72 ◦C for 10 min. All samples included in this study were sequenced, using an ABI 3130 DNA sequencer.

Statistical analysis

The OpenEpi software (https://www.openepi.com/TwobyTwo/TwobyTwo.htm) was implemented to analyze the allele and genotype data of the human samples studied.

Analysis of the SMAD9 (GCC)-repeat across vertebrates

The interval between +1 and +100 of the TSS of the SMAD9 was searched across all species in which SMAD9 was annotated, based on Ensembl 104. The Ensembl alignment program was implemented for the sequence alignments across the selected species.

Predicting RNA-RNA Interactions

The RNAstructure software ²⁶ was implemented to study example RNA/RNA dimerization patterns and potential accessibility variations across those dimers.

Allele frequency compartment

The SMAD9 (GCC)-repeat allele frequency compartment was significantly skewed in the NCD group vs. controls.

We detected two predominantly abundant alleles of 7 and 9-repeats in the human subjects studied in both groups (Table 1, Fig. 1A). At significantly lower frequencies, we detected repeats of 8, 10, and 11, of frequencies < 0.03. The allele frequency compartment was significantly skewed in the NCD group vs. controls, mainly due to the excess of 7 vs. 9-repeats in the NCD group (Yates corrected p = 0.009).

Table 1

Alleles and genotypes detected at the human *SMAD9* (GCC) and (GCT)-residue in the NCD and control groups.
Alleles
(GCC)-repeat	Control	NCD
7	150	182
8	1	3
9	171	141
10	10	8
11	2	0
	334	334
(GCT)-residue	Control	NCD
1	125	108
2	209	225
3	0	1
	334	334

A (GCT)-residue was detected at the immediate downstream sequence of the (GCC)-repeat (Table 1, Fig. 1B). While (GCC)7 was in linkage disequilibrium (LD) with (GCT)2, (GCC)9 and other rarer alleles of the (GCC)-repeat were detected in conjunction with (GCT)1 and (GCT)2. At the (GCT)-residue, (GCT)1 and (GCT)2 formed 99.9% of the allele pool in the human samples studied, and the allele frequency compartment was not significantly different at this residue between NCDs and controls.

Genotype compartment

The (GCC)-repeat genotype compartment was skewed in the NCD vs. control groups, whereas the (GCT)-residue genotype compartment was not significantly skewed between the two groups.

In the (GCC)-repeat compartment, genotypes consisting of (GCC)7 were significantly enriched in the NCD group, whereas genotypes consisting of (GCC)9 were significantly enriched in the control genotype compartment (Yates corrected p = 0.01). The (GCT)-residue genotype compartment was not significantly different between the two groups (Table 2, Fig. 2).

Table 2

Combinatory genotypes of the *SMAD9* (GCC) and (GCT) residue (order of numbers for each repeat represents their haplotypic position, i.e., 7/7 + 2/1 represents two haplotypes: 7 − 2 and 7 − 1).
Combinatory Genotypes
(GCC)n + (GCT)-residue	Controls	NCDs
7/7 + 2/1	0	1
7/7 + 2/2	39	51
7/7 + 2/3	0	1
7/8 + 2/1	0	2
7/9 + 2/1	48	48
7/9 + 2/2	20	23
7/10 + 2/2	4	2
7/10 + 2/1	0	1
8/9 + 1/1	1	0
8/10 + 2/1	0	1
9/9 + 1/1	28	20
9/9 + 2/1	11	11
9/9 + 2/2	9	4
9/10 + 1/1	2	0
9/10 + 2/2	2	0
9/11 + 1/2	2	0
10/10 + 1/1	1	2
	167	167

Identification of five genotype combinations in the NCD group only.

We found five combinatory genotypes that were detected in the NCD group and not in the controls (Table 2, Fig. 3A). Those genotypes were enriched with (GCC)7 and lacked (GCC)9, and encompassed 6 out of 167 patients studied (Mid-p exact = 0.007) (Table 3). Those patients received possible diagnoses of cerebrovascular disease (CVD) and AD. Of note, while (GCC)7 was in LD with (GCT)2 in all the control individuals studied, (GCC)7-(GCT)1 and (GCC)7-(GCT)3 were detected in the NCD group only.

Table 3

NCD patients harboring disease-only combinatory genotypes.
Patient No.	Gender	Age	AMTS	MMSE	Combinatory Genotypes	Possible Diagnosis
1	F	86	4	12	8/10 + 2/1	AD
2	F	83	3	1	7/8 + 2/1	AD + CVD
3	F	64	2	NA	7/8 + 2/1	CVD
4	F	76	1	10	7/7 + 1/2	CVD
5	M	74	5	NA	7/7 + 2/3	AD
6	F	66	4	16	7/10 + 2/1	CVD
AMTS = Abbreviated mental test score
MMSE = Mini-Mental State Exam
AD = Alzheimer’s disease
CVD = Cerebrovascular disease
NA = Not available

Identification of four combinatory genotypes in the controls only.

In contrast to the NCD-only genotypes that were enriched with (GCC)7 and lacked (GCC)9, the control-only genotypes exceptionally consisted of (GCC)9, and lacked (GCC)7. Those genotypes encompassed 7 out of 167 controls studied (Mid-p exact = 0.003) (Table 2, Fig. 3B).

SMAD9 (GCC)-repeat expanded specifically in primates.

Across all the vertebrate species studied, the SMAD9 (GCC)-repeat specifically expanded in primates (Fig. 4), and reached maximum length in human.

Here, we report a novel locus for late-onset NCD and indication of natural selection at a (GCC)-repeat in the 5′ UTR of the human SMAD9 gene. This locus may convey genotypes that specifically (unambiguously) predispose or protect against moderate to severe late-onset NCD in human. The NCD patients harboring the specific genotypes encompassed a spectrum of possible diagnoses, including CVD and AD.

The primary importance of (GCC)-repeats stems from a possible link between that type of STR and natural selection, mainly for two reasons: Firstly, (GCC)-repeats are specifically enriched in the exons. Secondly, CpG-rich sequences are mutation hotspots ²⁷, and frequently interrupted by single nucleotide substitutions as a result of C to T transitions, which is also the likely possibility at the (GCT)-residue at the immediate downstream flanking sequence of human SMAD9 (GCC)-repeat. Expansion of the SMAD9 (GCC)-repeat in primates, and not in any other order, supports selective advantage of this repeat in this order.

We found significant excess of the (GCC)7 allele in the NCD group and genotypes that consisted of (GCC)7 and not (GCC)9 in this group only. On the contrary, we found genotypes in the control group only and not in the NCDs, that consisted of (GCC)9 and not (GCC)7. Based on the above findings, we propose that the (GCC)7 allele may function as risk factor for late-onset NCD, whereas (GCC)9 may be protective. Similar to our findings in SMAD9, we have previously reported another predominantly biallelic (GCC)-repeat of 8 and 9 repeats in the 5′ UTR of the human SBF1 gene, in which excess of the shorter allele was detected in the NCD group ²⁸.

Searching the Genome Aggregation database (gnomAD) for the human SMAD9 (GCC)-repeat yielded inconclusive data for the annotated alleles and genotypes (https://gnomad.broadinstitute.org). The above finding is most likely due to the frequent failure of the general whole-exome sequencing methods to capture GC-rich sequences. Successful PCR amplification of the human SMAD9 gene is challenging, and warrants stringent conditions and special GC-rich buffer preparations as described in the Methods. Furthermore, this imperfect STR, which is disrupted by T nucleotides in its 3′ end, as revealed by the (GCT)-residue, indicates that conventional fragment analysis may not be an efficient method for scoring (GCC)-repeats. The above necessitates sequencing of every sample included in the study for obtaining accurate data.

SMAD9 is predominantly expressed in the brain and skeletal tissues ^18,29,30, and the protein encoded by this gene can translocate into the nucleus and affect transcriptional regulation of target genes. Higher order brain functions and skeletal phenotypes (characteristics that have significantly diverged in primates vs. other orders of animals) may be selection forces for the expansion of this STR in primates. Skewed genetic architecture in the group of NCD individuals with moderate to severe dysfunction of brain functions in our study, reflected predominantly in the AMTS and MMSE tests, supports a role for this STR in the human higher brain functions. Various (GCC/GGC)-repeats of the similar length range to the human SMAD9 gene STR can alter gene expression activity ^31,32. Our bioinformatics analysis revealed that the number of (GCC)-repeats may change the RNA secondary structure (stem-loops) and accessibility (unpaired RNA bases) of, at least, exons 1 and 2 of human SMAD9 (Fig. 5). RNA stem-loops in structurome data reveals widespread association with protein binding sites ³³, which may, in turn, alter the processes linked to transcription and translation.

Another interesting feature at this locus and a number of other previously reported instances is the low frequency alleles, which might have been subject to negative natural selection ^8,14,34. In human SMAD9, examples of those alleles are (GCC)8 and (GCC)10. Two genotypes consisting of those rare alleles i.e., 7/8 and 8/10, were detected in the NCD group only. Genotypes consisting of low frequency alleles at a (GCC) locus in the NCD patients were also detected in the RASGEF1C and SBF1 gene loci ^14,28. While allele and genotype-wise, the (GCT)-residue did not skew in the NCD group vs. controls, conjunction of (GCT)1 and (GCT)3 with (GCC)7 were detected in two NCD patients, and not in any controls.

Reported instances of STR allelic natural selection at non-coding loci in human are rare ^8,14, and the SMAD9 (GCC)-repeat provides a potentially valuable locus to further test this phenomenon.

It is warranted that this STR locus is sequenced in larger samples and in a spectrum of neurological and skeletal disorders. Mechanisms underlying allele and genotype selection should also be examined in the future functional studies.

We propose natural selection and a novel genetic locus for late-onset NCD in the SMAD9 5′ UTR (GCC)-repeat in human. This locus contains genotypes that may specifically link to or protect against late-onset NCD, a feature that may enhance the perspective of disease pathogenesis in disorders that appear to be complex, and yet may be linked to unambiguous genotypes at certain STR loci. It should be noted that this is a pilot study, warranting replication by independent studies.

AD: Alzheimer’s disease

AMTS: Abbreviated Mental Test ScoreC

CVD: Cerebrovascular disease

LD: Linkage disequilibrium

MMSE: Mini-Mental State Exam

SMAD9: SMAD Family Member 9

STR: Short tandem repeat

TSS: Transcription start site

UTR: Untranslated region

Author Contributions Statement

Samira Alizadeh and Safoura Khamse performed the molecular experiments and allele and genotype analyses. Hossein Afshar collected the human samples and their clinical information. Hamid Reza Khorram Khorshid and Ahmad Delbari contributed to data collection and coordination. Mina Ohadi conceived and supervised the project, and wrote the manuscript.

Data Availability

The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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You are reading this latest preprint version

A primate-specific (GCC) repeat in the untranslated region of SMAD9 undergoes natural selection and links with late-onset neurocognitive disorder in human

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Abstract

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Introduction

Materials And Methods

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Competing Interests

Status:

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