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Global Research journal of Natural Science

& Technology (GRJNST)

Volume: 04 - Issue 3 (2026), 2083

ISSN P: 2790-7643 ISSN E: 2790-7651

www.grjnst.net

https://doi.org/10.53762/grjnst.04.03.04

Mutational Analysis of TRAPPC9 IN Non-syndromic Intellectual Disability

in Selected Patients

Received: 28 March 2026. Accepted: 20 April 2026. Published: 8 May 2026

Mubara Mubashar

Department of Zoology,

Lahore College for Women University, Lahore, Pakistan

Farva Razzaq Maham

Department of Chemistry,

Superior Campus for University Programs, Mandi Bahauddin, Pakistan

Saman Mumtaz

Department of Zoology,

Lahore College for Women University, Lahore, Pakistan

Farooq Ahmad

Sustainable Development Study Centre, GC University Lahore, Pakistan

Fizza Hassan

Sustainable Development Study Centre, GC University Lahore, Pakistan

Laraib Saleem

Department of Zoology,

Lahore College for Women University, Lahore, Pakistan

Hafiza Komal Naeem

Department of Agriculture, University of Florence, Italy

Corresponding Author Email: fagondal82@gmail.com

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Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use and distribution

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Abstract

Intellectual incapacity (identity) is a neurodevelopmental circumstance

characterized through good sized obstacles in cognitive functioning and

adaptive behavior, with onset before the age of 18. It is usually diagnosed in

early formative years due to developmental delays in motor, cognitive, and

speech abilities, and is generally described by way of an IQ under 70. Identity is

classed into syndromic and non-syndromic kinds and further divided into mild,

mild, extreme, and profound categories based totally on severity. This takes a

look at centered on non-syndromic identification patients, regarding medical

evaluation and genetic evaluation. Blood samples have been accrued, and DNA

became analyzed the usage of PCR with TRAPPC9 gene primers, followed by

gel electrophoresis and sequencing. No mutation became detected in exon 14 of

the TRAPPC9 gene, suggesting the want for broader genetic investigations to

higher understand and help reduce the prevalence of intellectual incapacity.

Keyword: Mutation, Syndrome, Neurodevelopment, Patients, Cognitive

behavior

INTRODUCTION

Intellectual disability (identity) is a neurodevelopmental sickness characterized through

tremendous obstacles in cognitive functioning and adaptive behavior, with onset before

18 years of age, affecting about 1.five–2% of the populace. it is generally identified in

childhood because of developmental delays, even though formal analysis is based on an

IQ underneath 70. Individuals with id are at a higher danger of intellectual health issues

as compared to their generally growing peers. Identification is assessed into syndromic

and non-syndromic bureaucracy; syndromic identification includes additional bodily or

metabolic abnormalities, while non-syndromic id includes isolated cognitive impairment.

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Each genetic and environmental elements make a contribution to id, along with

chromosomal abnormalities and monogenic mutations (X-connected and autosomal)

(Marangi et al., 2013).

Consanguinity, particularly well-known in countries like Pakistan, drastically will

increase the prevalence of autosomal recessive highbrow disability. Neurodevelopmental

problems regularly proportion overlapping symptoms and genetic mechanisms, related to

pathways consisting of protein synthesis, transcriptional regulation, and synaptic

signaling. some of the genes implicated, TRAPPC9 encodes the NIBP protein, which

performs a crucial role in neurogenesis, synaptic plasticity, and myelination via NF-κB

signaling. Mutations in TRAPPC9 are associated with non-syndromic identification and

may present with functions including microcephaly, speech impairment, and brain

abnormalities (Abbasi et al., 2017).

Intention was to analyze mutations inside the TRAPPC9 gene in patients with non-

syndromic intellectual disability. Basic goals was to perceive the genetic foundation of

highbrow impairment throughout age agencies, increase awareness about inherited

genetic problems, and explore capability novel gene mutations.

METHODOLOGY

Affected individuals were clinically evaluated with the assistance of qualified

psychiatrists. Detailed assessments were conducted to identify cognitive, behavioral, and

developmental abnormalities associated with intellectual disability (ID). Clinical history,

including developmental milestones, speech development, and behavioral patterns, was

recorded. This evaluation helped in confirming the diagnosis of intellectual disability

and distinguishing between syndromic and non-syndromic cases.

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Families with more than three affected individuals were identified from the district of

Mandi Bahauddin, Punjab. Field visits were conducted to collect detailed family

histories and demographic information. Pedigree charts were constructed for each family

to analyze inheritance patterns and identify possible modes of transmission, particularly

focusing on autosomal recessive inheritance due to high consanguinity rates. Only

families meeting the inclusion criteria were enrolled in the study.

Prior to sample collection, informed consent was obtained from all participants or their

guardians. Approximately 5–10 ml of peripheral blood was collected from each affected

individual using sterile techniques. Blood samples were collected in EDTA-containing

falcon tubes to prevent coagulation. The samples were properly labeled and transported

to the laboratory under controlled conditions. For preservation, the samples were stored

at −20°C until further processing.

Genomic DNA was extracted from blood samples using a standard protocol followed at

LCWU. The extraction process involved cell lysis to release cellular contents, followed

by digestion of proteins using proteinase K. Proteins were precipitated using saturated

sodium chloride (NaCl), and DNA was subsequently precipitated using isopropanol.

This method ensured the isolation of high-quality DNA suitable for downstream

molecular analysis.

Day 1: Cell Lysis and Washing

Frozen blood samples were thawed at room temperature using tap water. Tris-EDTA

(T.E) buffer was added in two steps to ensure proper mixing and cell suspension. The

samples underwent multiple washing steps followed by centrifugation at 3000 rpm for

25–30 minutes at 25°C. After each centrifugation, the supernatant was discarded, and

the pellet was resuspended. This washing process was repeated three to four times to

remove impurities and obtain a clean cell pellet.

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After the final wash, TNE buffer was added to dissolve the pellet, followed by the

addition of 10% SDS and proteinase K to facilitate protein digestion. The samples were

incubated overnight at 37°C in a shaking incubator to ensure complete lysis and

digestion.

Day 2: Protein Removal and DNA Precipitation

On the second day, samples were checked for complete digestion. Chilled NaCl was

added to precipitate proteins, followed by incubation in the freezer. A phenol-

chloroform-isoamyl alcohol (PCI) solution was then added, and samples were gently

mixed. Centrifugation was performed to separate the layers, and the upper aqueous layer

containing DNA was carefully transferred to new tubes.

Equal volumes of isopropanol were added to precipitate DNA, which appeared as visible

threads. The DNA was then centrifuged, and the pellet was washed with 70% ethanol

to remove impurities. After drying, the DNA pellet was dissolved in low TNE buffer

and incubated overnight to ensure complete dissolution.

Day 3: DNA Stabilization and Storage

On the third day, samples were treated to inactivate nucleases by incubating at 65–70°C

in a water bath. After cooling, DNA samples were transferred into labeled screw-cap

tubes and stored in duplicate at −20°C. DNA concentration and integrity were later

assessed using agarose gel electrophoresis.

DNA Quantification

DNA concentration and quality were determined using agarose gel electrophoresis. A

0.8% agarose gel was prepared using TBE buffer, and ethidium bromide was added for

DNA visualization under UV light. DNA samples mixed with loading dye were loaded

into wells, and electrophoresis was performed for 25–30 minutes. The gel was visualized

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using a gel documentation system, and DNA concentration was estimated by comparing

band intensity with known DNA standards. This method also allowed assessment of

DNA integrity.

Polymerase Chain Reaction (PCR)

PCR was performed to amplify specific regions of the TRAPPC9 gene prior to

sequencing. The reaction mixture was prepared using extracted genomic DNA, gene-

specific primers, nucleotides (dNTPs), buffer, and Taq DNA polymerase.

PCR Conditions

PCR amplification was carried out using a thermal cycler with the following conditions:

 Initial denaturation at 94°C for 5 minutes

 Denaturation at 94°C for 30 seconds

 Annealing at 59°C for 30 seconds

 Extension at 72°C for 30 seconds

 Final extension at 72°C for 10 minutes

These steps were repeated for multiple cycles to ensure sufficient amplification of the

target DNA region.

Denaturation

During denaturation, the double-stranded DNA was separated into single strands by

breaking hydrogen bonds at high temperatures (94–98°C). This step is critical for

allowing primers to bind to the DNA template in subsequent steps.

Annealing

In the annealing step, primers specific to the TRAPPC9 gene bind to complementary

sequences on the single-stranded DNA templates. The temperature (59°C) was

optimized based on primer characteristics to ensure specificity and efficient binding.

Extension

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During extension, Taq DNA polymerase synthesized new DNA strands by adding

nucleotides complementary to the template strand at 72°C. This step resulted in the

amplification of the target DNA region.

Pre-Sequencing Preparation

Following PCR amplification, the products were verified through gel electrophoresis to

confirm successful amplification. The PCR products were then purified and prepared

for sequencing to identify potential mutations in the TRAPPC9 gene.

Reaction mixture for Pre-sequencing PCR

The PCR reaction mixture was prepared in a total volume of 15 μl. It consisted of 2 μl

of genomic DNA with a concentration of 50–60 ng/μl. To this, 0.5 μl of forward

primer and 0.5 μl of reverse primer (each at 10 nmol concentration) were added.

Additionally, 5 μl of master mix was included in the reaction, along with 7 μl of

injection water to make up the final volume.

Primer sequence of TRAPPC9 Gene

The primer sequences for exon 14 of the TRAPPC9 gene were designed as follows: the

forward primer (F.P) sequence was 5′-GAAGAGGAGCCCCGTACTCT-3′, and the

reverse primer (R.P) sequence was 5′-GTGACCCTCGTGCACACTAC-3′. These

primers were used to amplify a product with a size of 247 base pairs (bp).

RESULTS

Ethical approval for this study was obtained from the Ethical Committee of Lahore

College for Women University (LCWU), Lahore, Pakistan. Intellectually disabled

families were identified through field visits in Mandi Bahauddin, where affected

individuals were clinically assessed with the assistance of psychologists. Diagnosis was

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based on factors including speech disability, motor delay, age, gender, height, weight,

prenatal and postnatal history, and IQ level. Behavioral characteristics such as mood,

speech patterns, eating habits, sleep, attention, and additional traits like aggression,

hyperactivity, impulsivity, and tantrums were also recorded.

Two families with intellectual disability were identified, and one family (PKNSID0091)

was selected for molecular analysis. Blood samples were collected with informed consent

and stored at −20°C. Pedigree analysis was performed using Cegat software, indicating

an autosomal recessive inheritance pattern associated with consanguinity. DNA was

extracted, followed by PCR amplification using TRAPPC9 gene primers. Gel

electrophoresis confirmed the amplified products, and DNA sequencing was carried out

to detect potential mutations.

The selected family, from Mandi Bahauddin, showed a history of cousin marriages and

included three affected individuals across three generations. The pedigree suggested

autosomal recessive inheritance, with unaffected parents and affected offspring. All

affected members exhibited non-syndromic intellectual disability.

Table 1. History of affected family PKNSID0091

Family ID

Gender

PKNSID0091

Male

Age

25 years

70kg

Weight

IQ Level

Severe

No. of affected 03

individuals

At

birth

time Birth trauma

Neonatal (1^stfour Fitz after birth

condition

weeks of life)

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Table 2. Signs of disorders in the patient with intellectual disability

Signs

Yes

No

✓

Floppy Limbs

Problem in feeding

Cleft Lip

Weak Limbs

Club feet

Lump on back

Lump at navel

Child has serious delays in

sitting

✓

Child has serious delays in

standing

✓

Child has serious delays in

walking

Child has difficulty in

seeing in daytime

Child has difficulty in

seeing at night

✓

Child appears to have

difficulty in hearing

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Child has difficulty in

understanding

✓

Child has difficulty in

moving arms

✓

Child loses consciousness at

sometimes

Child cannot name objects

like toys, books etc

Child is not learning to do

things like other children

Sequencing results were analyzed using Chromas software to visualize and extract DNA

sequences obtained from Sanger sequencing. The reference sequence of the TRAPPC9

gene was retrieved from the Ensemble Genome Browser, including both exons and

introns. Sequence alignment was performed using NCBI BLAST by comparing the

obtained (query) sequence with the reference (subject) sequence in FASTA format. The

query sequence was copied from Chromas and aligned against the reference gene

sequence to identify variations.

The analysis of family PKNSID0091 revealed no mutation in exon 14 of the

TRAPPC9 gene. Further analysis is ongoing to investigate potential mutations in other

exons of the gene.

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Figure 1. Sequencing chromatogram of exon 14 of TRAPPC9

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Figure 2. No mutation in exon 14 of family PKNSID0091

Figure 3. Pedigree of family PKNSID0090

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Figure 4. Pedigree of family PKNSID0091

DISCUSSION

Intellectual disability (ID) is a neurodevelopmental disorder characterized by significant

limitations in cognitive functioning and adaptive behavior, with onset before 18 years of

age. It affects approximately 1.5–2% of populations in Western countries and is usually

diagnosed when IQ is below 70, although early identification often occurs due to

developmental delays in motor, speech, and cognitive skills. Individuals with ID have a

higher risk of mental health disorders compared to typically developing individuals. ID

is classified into mild, moderate, severe, and profound categories, and may be syndromic

or non-syndromic in nature. (Mefford et al., 2012).

Globally, consanguinity is widely practiced, particularly in South Asia, Africa, and the

Middle East, with Pakistan showing a high rate (~65%). This increases the prevalence

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of autosomal recessive intellectual disability. In Pakistan, moderate ID prevalence is

approximately 65/1000 and severe ID about 19/1000. Increased consanguinity

contributes significantly to genetic disorders, including non-syndromic ID (De Ligt et

al., 2012).

The present study focused on molecular analysis of families with non-syndromic

intellectual disability from Mandi Bahauddin, Punjab. Pedigree analysis revealed an

autosomal recessive inheritance pattern. One family (PKNSID0091) with three affected

individuals was selected. Blood samples were collected at LCWU, DNA was extracted,

and gel electrophoresis confirmed DNA presence. PCR amplification was performed

using TRAPPC9 gene primers (Ibrahim et al., 2023).

The TRAPPC9 gene, located on chromosome 8, is involved in NF-κB signaling,

neuronal development, and intracellular protein trafficking. It plays an important role in

neurogenesis and brain development. Although mutations in TRAPPC9 have been

associated with intellectual disability, no pathogenic alteration was identified in this

study. Its exact functional role in neuronal impairment remains unclear, particularly

whether dysfunction is due to gene mutation or downstream protein effects (Wilton et

al., 2020).

Figure 5. TRAPPC9 Gene on Chromosome 8

https://chromodisorder.org/latest-research-articles/chromosome-8p-abnormalities-

studied-in-a-group-of-individuals/

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In Pakistan, cousin marriage is culturally common and is associated with a higher

incidence of genetic disorders. Consanguineous families provide a valuable resource for

identifying novel genes due to their unique genetic background. In this study, family

PKNSID0091 was selected for molecular analysis, and TRAPPC9 gene primers were

used for PCR amplification. However, sequencing results showed no mutation in the

analyzed region. This suggests that mutations may be present in other exons of the same

gene or that other genes may be responsible for the disorder in this family.

CONCLUSION

This research revealed that there is no mutation detected on the TRAPPC9 gene at exon

14 of ID patient. So, by this research it is concluded that may be some other exon or

gene is having mutation for the family PKNSID0091 or we can say that may be some

allelic heterogeneity present in them.

REFERENCES

Abbasi, A. A., Blaesius, K., Hu, H., Latif, Z., Picker‐Minh, S., Khan, M. N., ...

andKaindl, A. M. 2017. Identification of a novel homozygous TRAPPC9 gene

mutation causing non‐syndromic intellectual disability, speech disorder, and

secondary microcephaly. American Journal of Medical Genetics Part B:

Neuropsychiatric Genetics, 174(8): 839-845.

Alvarez-Mora, M. I., Corominas, J., Gilissen, C., Sanchez, A., Madrigal, I., and

Rodriguez-Revenga, L. 2021. Novel compound heterozygous mutation in

TRAPPC9 gene: the relevance of whole genome sequencing. Genes, 12(4): 557.

Amin, M., Vignal, C., Eltaraifee, E., Mohammed, I. N., Hamed, A. A., Elseed, M. A.,

... andDorboz, I. 2022. A novel homozygous mutation in TRAPPC9 gene causing

GRJNST, Volume: 04 - Issue 3 (2026) / ISSN P: 2790-7643

Article ID: 2083

https://doi.org/10.53762/grjnst.04.03.04

G. 2083

Page 16

autosomal recessive non-syndromic intellectual disability. BMC Medical

Genomics, 15(1): 1-5.

Bessa, C., Lopes, F., andMaciel, P. 2012. Molecular genetics of intellectual disability.

Chelly, J., Khelfaoui, M., Francis, F., Chérif, B., and Bienvenu, T. 2006. Genetics and

pathophysiology of mental retardation. European Journal of Human Genetics, 14(6):

701-713.

Chen, C., Chen, D., Xue, H., Liu, X., Zhang, T., Tang, S., ... & Xu, X. (2018).

IDGenetics: a comprehensive database for genes and mutations of intellectual

disability related disorders. Neuroscience Letters, 685, 96-101.

Eggermann, T., Perez de Nanclares, G., Maher, E. R., Temple, I. K., Tümer,

Z., Monk, D., Mackay, D. J., Grønskov, K., Riccio, A., Linglart, A., and Netchine,

I. 2015 Imprinting disorders: a group of congenital disorders with overlapping

patterns of molecular changes affecting imprinted loci. Clin Epigenetics 7, 123.

Forster, S., Gray, K. M., Taffe, J., Einfeld, S. L., andTonge, B. J. 2011. Behavioural

and emotional problems in people with severe and profound intellectual

disability. Journal of Intellectual Disability Research, 55(2); 190-198.

Hnoonual, A., Graidist, P., Kritsaneepaiboon, S., andLimprasert, P. 2019. Novel

compound heterozygous mutations in the TRAPPC9 gene in two siblings with

autism and intellectual disability. Frontiers in Genetics, 10, 61

Ibrahim, N., Naz, S., Mattioli, F., Guex, N., Sharif, S., Iqbal, A., ... and Reymond, A.

2023. A Biallelic Truncating Variant in the TPR Domain of GEMIN5 Associated

with Intellectual Disability and Cerebral Atrophy. Genes, 14(3): 707

Kanwal, M., Alyas, S., Afzal, M., Mansoor, A., Abbasi, R., Tassone, F., and Mazhar,

K. 2015. Molecular diagnosis of fragile X syndrome in subjects with intellectual

GRJNST, Volume: 04 - Issue 3 (2026) / ISSN P: 2790-7643

Article ID: 2083

https://doi.org/10.53762/grjnst.04.03.04

G. 2083

Page 17

disability of unknown origin: implications of its prevalence in regional

Pakistan. PLoS One, 10(4): e0122213.

Karam, S. M., Riegel, M., Segal, S. L., Félix, T. M., Barros, A. J., Santos, I. S., ... abd

Black, M. 2015. Genetic causes of intellectual disability in a birth cohort: A

population‐based study. American Journal of Medical Genetics Part A, 167(6):

1204-1214

Kaufman, L., Ayub, M., and Vincent, J. B. 2010. The genetic basis of non-syndromic

intellectual disability: a review. Journal of neurodevelopmental disorders, 2(4): 182-

209.

Khan, M. A., Khan, S., Windpassinger, C., Badar, M., Nawaz, Z., and Mohammad,

R. M. 2016. The molecular genetics of autosomal recessive nonsyndromic

intellectual disability: a mutational continuum and future recommendations. Annals

of Human genetics, 80(6): 342-368.

Marangi, G., Leuzzi, V., Manti, F., Lattante, S., Orteschi, D., Pecile, V., ...

andZollino, M. 2013. TRAPPC9-related autosomal recessive intellectual disability:

report of a new mutation and clinical phenotype. European Journal of Human

Genetics, 21(2): 229-232.

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