PJZ_58_4_1643-1651

INTRODUCTION

Ocular albinism (OA) and oculocutaneous albinism (OCA) are the two categories of albinism. In former case, hypopigmentation affects only eyes, whereas, in later case, hypopigmentation occurs in hair, skin and eyes. Globally, OCA is the most common disorder that is distributed between various ancient, linguistic and cultural groups (Hamid et al., 2018). It is reported that albinism appears in 1 in 17,000 to 1 in 20,000 people, which suggests that 1 person in 70 people is the carrier of OCA-mutated gene. OCA follows autosomal recessive pattern of inheritance, therefore consanguinity is the major contributing factor (Summers, 2009). Up till now, eight types of OCA have been reported. Clinically, all the reported types show variable clinical features (Garcia Galvão et al., 2019).

OCA1 has two subtypes i.e. OCA1A and OCA1B. OCA1A is the most common type of albinism caused by mutation in TYR gene, located on chromosome 11q14.3 (Simeonov et al., 2013). Besides OCA1A, the subtype OCA1B is a mild to moderate form of OCA1 (Arshad et al., 2018; Norman et al., 2017). OCA type 2 is caused due to mutation in OCA2 gene that is located on chromosome 15 (Preising et al., 2007). OCA3 is the third type of OCA in which TYRP1 gene is mutated. This gene is located on chromosome 9p23. OCA4 is associated with mutations in SLC45A2 gene, which is present on 5p13 (Inagaki et al., 2006). OCA5 is associated with locus on chromosome 4q24; however the specific defective gene has not been reported yet (Kausar et al., 2013). Mutations in the SLC24A5 gene (solute carrier family-24, member 5) lead to the appearance of OCA6. This gene is located at 15q21.1 (Grønskov et al., 2013). C10orf11 mutations are linked to the OCA7 type of albinism, whose responsible gene is located on chromosome 10q22.2-q22.3 locus. OCA8 is a novel type of OCA that is recently discovered to be associated with mutations in DCT on chromosome 13q31-q32 (Garrido et al., 2021). Most of the reported genes are involved in melanogenesis. Previous research has reported the presence of TYR, OCA2, TYRP1, SLC45A2, and SLC24A5 genes among the others in Pakistani population (Gul et al., 2019; Kausar et al., 2013; Morice-Picard et al., 2014; Shah et al., 2014a; Shahzad et al., 2017; Zhang et al., 2019).

The present study is designed to explore the genetic basis of OCA in two affected families from Azad Jammu and Kashmir. This region is located between the borders of India and Pakistan. Studies from this area revealed that mutations in TYR and OCA2 genes are the major causative variants in OCA families (Konno et al., 2009; Shah et al., 2014b; Shahzad et al., 2017).

MATERIALS AND METHODS

Two families were recruited from different areas of Azad Jammu and Kashmir. For the extraction of genomic DNA, blood samples were taken from the affected as well as normal members. Interviews were conducted from all the available members of families at the time of enrollment. Pigmentation in the hair, skin and eyes was documented by taking photographs of the affected members.

Whole exome sequencing

Genomic DNA of affected members (IV:6) of family A and V:7 of family B were subjected to whole exome sequencing for the identification of responsible variants of OCA. Whole exome sequencing library enrichment was done with the Roche Nimble-Gen SeqCap EZ Exome v3 kit. Then, Illumina HiSeq 4000 was applied to sequence the selected samples. With the sequencing reads kept at 36x, each read from the target area covered 94% of the average means depth.

After removing adaptors and low-quality reads, the qualifying raw fastq file was processed in accordance with the “Best Practices for Germline SNP and Indel Discovery” published by GATK. The pure sequences were aligned using Burrows-Wheeler Aligner (BWA-MEM v0.7) to the UCSC Genome Browser hg19. 12), and Picard v1.141 was used to remove PCR duplicates. No data containing synonymous, noncoding variations with a minor allele frequency (MAF) of 0:01 is included in the research from the exome aggregation consortium (ExAC), 1000 genomes, or genome aggregation database (gnomAD) (https://gnomad .broadinstitute.org/) databases. Only segments that alter a gene’s splice site’s coding regions, synonymous splice site variations, nonsense segments, and non-synonymous segments were considered for interpretation. Additionally, a range of in silico algorithms, such as SIFT (http://sift. PolyPhen2) (http://genetics.bwh.org), jcvi.org/., mutation taster (http://www.mutationtaster.org/), FATHMM (http://fathmm.biocompute.org.uk/), and Harvard University (http://harvard.edu/pph2/) were utilized to assess potential negative impacts of pathogenic variants on the structure and function of proteins.

Variant validation and segregation testing

Sanger sequencing was used to confirm variant zygosity in all available family members. After filtering, remaining candidate variants were amplified by PCR using primers designed with Primer3 software (Untergasser et al., 2012). Bidirectional sequencing of PCR products was performed using standard protocols. Chromas version 2.6.6 and Alamut Visual Plusversion 1.6.2 were used to analyze DNA sequences.

Protein 3D modelling and docking

For designing 3D protein models of normal and mutant TYR protein and its close functional interactor DCT protein, I-TASSER online (Yang et al., 2015) tool was used. 3D protein model with highest confidence score (C-Score) was chosen for additional exploration (Muzammal et al., 2022). Designed 3D models were visualized by Chimera 1.13.1 (Pettersen et al., 2004). After designing and visualizing 3D protein models, protein-protein docking was carried out using online cluspro server tool with its close functional interactors (Kozakov et al., 2017). Close functional interactor was predicted via String Data base (Szklarczyk et al., 2019).

Multiple sequence alignment

To check the conservation of substituted amino acids in different species multiple sequence alignment was done using online tool Clustal Omega (Larkin et al., 2007; Sievers and Higgins, 2014).

RESULTS

Clinical manifestation

We recruited two consanguineous families affected with OCA from different regions of Azad Jammu and Kashmir. Affected individuals of both families showed clear signs and symptoms of OCA, including hypopigmentation in the eyes, white to pale hair color, reduced pigmentation in skin, impaired vision along with nystagmus, iris trans-illumination and photophobia.

Additionally, in family A, depigmentation was clearly observed in the skin, hair and eyes and impaired vision was reported along with continuous nystagmus. A summary of the clinical characteristics of the affected members of both families is shown in Table I.

Identification of pathogenic variants in OCA-affected families

Analysis of whole exome data of Family A identified previously reported mutation c. 62C>T: p.Pro21Leu in the exon 1 of TYR gene. Family B showed previously reported mutation c.832C>T, p.Arg278* in the TYR gene. Additionally, the segregation of these variations within their respective families is confirmed by Sanger sequencing.

In Family A, both the affected individual (IV: 5 and IV: 6) were homozygous mutant, while the normal individual (III: 5) was heterozygous carrier for the variant c.62C>T: p.Pro21Leu,while in Family B the normal individuals (IV:2,IV:3 and V:9) were heterozygous carrier for the variant c.832C>T, p.Arg278* and affected members (V: 6) and (V: 7) were homozygous mutant (Fig. 1).

Table I. Clinical findings of two Pakistani families showing mutations in TYR gene.

Family	Subject ID	Age (Years)	Sex	Mutation	Ethnolinguistic group	Hair color	Skin tone	Iris color
A	IV:3	54	F	c.62C>T: p.Pro21Leu	Syed	Black	Black	Black
	III:3	60	M			Black	Black	Black
	V:9	26	F			Black	Black	Black
	V:6	28	M			White	White	Brown
	V:7	30	M			White	White	Brown
B	III:2	47	F	c.1255G>A: p.Gly419Arg	Mughal	Black	Brown	Black
	III:1	50	M			Black	Brown	Black
	IV:3	23	M			White	White	Grey blue
	IV:4	26	M			White	White	Grey blue

Molecular modeling of normal and mutated TYR proteins

3D structure of wild-type and mutant (p. Pro21Leu) TYR proteins were predicted and superimposed to check the similarity (% identity) between two structures (wild-type and mutant (p. Pro21Leu) TYR proteins). The 3D structures of the wild-type and both mutant TYR proteins were overlaid using molecular modeling. The TYR protein wild-type mutant 2 (p. Pro21Leu) had the lowest similarity index of 82.42% (Fig. 2).

Mutant protein showed variable docking sites and docking residues when compared with wild-type TYR protein. These changes in the interacting residues showed the pathogenic effect of these variation in compound heterozygous state, which effect the interaction of TYR protein with its close interactor DCT protein. A study of the protein-protein interactions between the wild-type and both mutant TYR proteins, showed that the mutation caused a significant change in the docking locations, interacting residues, and bonding. Thirteen distinct residues and eighteen hydrogen bonds mediated the contact between the mutant p. Pro21Leu and the DCT protein (Fig. 3).

Multiple sequence alignment

Multiple sequence alignment of substituted amino acid were done and found that the substituted amino acids i.e. i.e. Pro21 is highly conserved throughout several species. Which confirms the importance of these amino acids. Conservation of substituted amino acids are shown in Figure 4.

DISCUSSION

Non-syndromic autosomal recessive OCA is a diverse group of disorders that affects 1 in 20,000 people worldwide. The incidence of the many subtypes of OCA varies significantly based on an individual’s ethnic background (Gargiulo et al., 2011; Kamaraj and Purohit, 2014). The most common symptom of OCA is a reduction in or absence of melanin synthesis, which leads to a partial or complete loss of skin, hair, and ocular pigmentation. Eight discrete autosomal genetic loci have already been identified to be associated with non-syndromic OCA. It is reported that OCA cases, including those in Pakistan, are primarily caused by mutations in the TYR (OCA1) and OCA2 (OCA2) genes (Gul et al., 2017). List of previously reported TYR gene mutations in Pakistan population are summarized in Table II.

The overall prevalence of TYR-associated pathogenic mutations in Pakistan is approximately 44%, while it is 46% in the European population (Jaworek et al., 2012). TYR pathogenic variant findings from other Asian ethnicities are inconsistent, but one regional subgroup of the Chinese population has over 70% of these reports, demonstrating considerable variation (Gul et al., 2017; Jaworek et al., 2012). A study on an Indian ethnic group revealed that individuals with OCA characteristics had a 60% incidence of TYR pathogenic mutations (Okamura et al., 2019; Sengupta et al., 2010). Furthermore, in other populations including Americans, Koreans, Japanese, and Italians, the OCA phenotype is mostly caused by the predominance of TYR pathogenic mutations (Hutton and Spritz, 2008).

With a frequency of about 23%, the pathogenic mutation of the TYR gene that is most frequently discovered in Pakistani OCA families is c.1255G>A; p.Gly419Arg, which is mainly displayed by Punjabi. Yet, OCA families from various linguistic groups, such as the Baloch, Saraiki, Sindhi, and Kashmiri language groups, have also been found to have a comparable pathogenic variation (Arshad et al., 2018; Gul et al., 2019; Hutton and Spritz, 2008; Ghodsinejad Kalahroudi et al., 2014; Shahzad et al., 2017). Another analysis also identifies the second most prevalent pathogenic variation, which accounts for around 21% of all known pathogenic variants: c.832C>T; p.Arg278*(Gul et al., 2019; Shakil et al., 2019). Moreover, c.1037-7T>A, a previously identified splice-site pathogenic mutation, has been found in a number of citizens with Pakistani origin (Gul et al., 2017, 2019; Shahzad et al., 2017).

Numerous research studies have documented novel, well-known, and founder mutations of the TYR gene. The most prevalent and common mutations in the population are c.832C>T: p.R278* and c.1255G>A: p.Gly419Arg variants of the TYR gene, which have been identified in 21 families (22.6%) and 20 families (23.8%), respectively. On the contrary, the founder mutations, such as c.230G>A (5.9%), are certain mutations that contribute frequently to the ancestry. About 90 variants, including 86.9% missense mutations, 7.1% nonsense mutations and the remainder changes causing splice site errors, deletions, and frame shifts, are connected to TYR-linked OCA in consanguineous families in Pakistan (Table II).

In this research, mutational analysis of two families, having non-syndromic OCA, was done by performing Sanger sequencing for the identification of underlying genes that cause disorder to appear. All families were depicting the symptoms of non-syndromic form of OCA. In exon 1 of the TYR gene, we discovered already reported pathogenic mutation c. 62C>T: p. Pro21Leu in the exon 1 of the TYR gene. Family B showed c.832C>T; p.Arg278* mutation in exon 2 of the TYR gene was discovered. As consanguineous marriages account for 60% of all marriages in Pakistan (Elahi et al., 1998), consanguinity is the major contributor for causing non-syndromic OCA in both families. The approach used here for the prediction of responsible gene and specific mutations is Sanger sequencing.

Table II. List of previously reported TYR gene mutations in Pakistani families associated with OCA.

No of families	Nucleotide change	Amino acid change	Mutation type/ Status	Ethnicity	Reference
1	c.62C>T	p.Pro21Leu	Missense/Homozygous	Punjabi	Kruijt et al. (2018)
1	c.1424G>A	p.Trp475*	Nonsense/Homozygous	Saraiki	Jaworek et al. (2012)
1	c.1231 T>C	p.Tyr411His	Missense/ Homozygous	Punjab	Shakil et al. (2019)
1	c.1217C>T	p. Pro406Leu	Missense/ Homozygous	Punjabi	Shakil et al. (2019)
1	c.1184+2 T>C	-	Splicing Error/ Heterozygous	Punjabi	Shakil et al. (2019)
1	c.115 Del 340-341	p.R278 *	Frameshift Nonsense	Pakistani	Gul et al. (2017)
1	c.1147 G>A	p.Asp383Asn	Missense/ Homozygous	Pakhtoon	Shakil et al. (2019)
1	c.1037-18 T>G	-	Splicing Error/ Heterozygous	Urdu speaking	Shakil et al. (2019)
4	c.1037-7 T>A	-	Splicing Error/ Heterozygous	Urdu Punjabi	Spritz et al. (1995)
1	c.1037G>T	p.Gly346Val	Missense/Homozygous	Punjabi	Shakil et al. (2019)
3	c.896A>G	p.Arg299His	Missense/Homozygous	Pakhtoon, Punjabi	Shakil et al. (2019)
1	c.593T>C	p.Ile198Thr	Missense/Homozygous	Pakistani	Shah et al. (2015)
1	c.585G>A	p.Trp195*	Nonsense/ Homozygous	Punjabi	Shakil et al. (2019)
1	c.943-948 deltcagct	p.315-316del	Deletion/Heterozygous	Punjabi	Shakil et al. (2019)
1	c.575C>A	p.Ser192Tyr	Homozygous	Kashmiri	Lee et al. (2021)
21	c.832C>T	p.Arg278*	Nonsense/ Homozygous	Urdu speaking, Punjabi, Pashtun	Konno et al. (2009), Sajid et al. (2021), Shahzad et al. (2017), Shakil et al. (2019), Spritz et al. (1995)
2	c.346C>T	p.Arg116*	Nonsense/ Homozygous	Saraiki, Punjabi	Gul et al. (2019)
1	c.895C>T	p.Arg299Cys	Missense/Homozygous	Saraiki	Jaworek et al. (2012)
1	c.715C>T	p.Arg239Trp	Missense/Homozygous	Kashmiri	Shahzad et al. (2017)
1	c.982G>C	p.Glu328Gln	Missense/Homozygous	Pakistani	(Lee et al. (2021)
2	c.240G>C	p.Trp80Cys	Missense/Homozygous	Pakhtoon	Arshad et al. (2018)
1	c.1037 G>A	p.Gly346Glu	Missense/Homozygous	Punjabi	Shakil et al. (2019)
1	c.826C>T	p.Cys276Arg	Missense/Homozygous	Pakhtoon	Konno et al. (2009)
1	c.308G>A	p.Cys103Tyr	Missense/Homozygous	Punjabi	Spritz et al. (1995)
1	c.272G>A	p.Cys91Tyr	Missense/Homozygous	Punjabi	Spritz et al. (1995)
3	c.132T.A	p.Ser44Arg	Missense/Homozygous	Kashmiri, Pakhtoon, Baloch	Arshad et al. (2018)
2	Exon 4-5 deletion	-	Deletion/Heterozygous	Punjabi	Shakil et al. (2019)
2	c.103T>C	p.Cys35Arg	Missense/Homozygous	Punjabi	Shah et al. (2014)
1	c.248T>G	p.Val83Gly	Missense/Homozygous	Pakhtoon	Shahzad et al. (2017)
4	c.230G>A	p.Arg77Gln	Missense/Homozygous	Kashmiri	(Shahzad et al. (2017)
1	c.1204C>T	p.Arg402*	Nonsense/Homozygous	Punjabi	Shakil et al. (2019)
1	c.223G>T	p.Asp75Tyr	Missense/Homozygous	Punjabi	Shakil et al. (2019)
1	c.164G>C	p.Cys55Ser	Missense/Homozygous	Punjabi	Shakil et al. (2019)
23	c.1255G>A	p.Gly419Arg	Missense/Homozygous	Punjabi, Sindhi, Pakhtoon, Kashmiri, Saraiki	Arshad et al. (2018), Shakil et al. (2022)

Molecular screening also revealed that all mutations are pathogenic. Besides this, all the families A and B showed homozygosity (Mut/Mut) in affected subjects. However, the carriers were heterozygous (WT/Mut).

CONCLUSION

In this study, we identified previously reported mutation in TYR gene in two families affected with non-syndromic OCA, from Azad Jammu and Kashmir, Pakistan. Our current findings and already reported data show that TYR gene is the causative gene in families affiliated with OCA in majority of cases in Pakistan. Moreover, research reported that all mutations are showing pathogenic status. This study expands the mutational spectrum of TYR gene in Kashmiri families.

Declarations

Acknowledgement

We are Also thankful to Higher Education Commission Pakistan (HEC) for awarding IRSIP Fellowship to conduct this study.

IRB approval

Informed consent was obtained from the participants prior to blood sampling. The study was also approved by the institutional review board (IRB) of Mirpur University of Science and Technology (MUST) Mirpur, AJK.

Ethical statement

The study was conducted according to the guidelines of the Declaration of Helsinki,and written informed consent was obtained from the participants prior to conduct the study.

Generative AI and AI-assisted technology statement

The authors declare that they have not used generative AI or AI-assisted technologies in the writing or editing of this manuscript.

Statement of conflict of interest

The authors have declared no conflict of interest.

REFERENCES

Arshad, M.W., Harlalka, G.V., Lin, S., D’Atri, I., Mehmood, S., Shakil, M., Hassan, M.J., Chioza, B.A., Self, J.E., Ennis, S., O’Gorman, L., Norman, C., Aman, T., Ali, S.S., Kaul, H., Baple, E.L., Crosby, A.H., Ullah, M.I. and Shabbir, M.I., 2018. Mutations in TYR and OCA2 associated with oculocutaneous albinism in Pakistani families. Meta Gene, 17: 48–55. https://doi.org/10.1016/j.mgene.2018.03.007

Elahi, M.M., Elahi, F., Elahi, A. and Elahi, S.B., 1998. Paediatric hearing loss in rural Pakistan. J. Otolaryngol., 27: 348–353.

Garcia Galvão, L.E., Tomaz, R. and de Sá Gonçalves, H., 2019. Terapia fotodinámica con luz de día en el tratamiento de queratosis actínicas en pacientes portadores de albinismo oculocutáneo: Presentación de 3 casos. Actas Dermo-Sifiliográf., 110: 407–410. https://doi.org/10.1016/j.ad.2018.02.035

Gargiulo, A., Testa, F., Rossi, S., Di Iorio, V., Fecarotta, S., de Berardinis, T., Iovine, A., Magli, A., Signorini, S., Fazzi, E., Galantuomo, M.S., Fossarello, M., Montefusco, S., Ciccodicola, A., Neri, A., Macaluso, C., Simonelli, F. and Surace, E.M., 2011. Molecular and clinical characterization of albinism in a large cohort of Italian patients. Invest. Opthalmol. Visual Sci., 52: 1281. https://doi.org/10.1167/iovs.10-6091

Garrido, G., Fernández, A. and Montoliu, L., 2021. HPS11 and OCA8: Two new types of albinism associated with mutations in BLOC1S5 and DCT genes. Pigment Cell Melanoma Res., 34(1): 10–12. https://doi.org/10.1111/pcmr.12929

Ghodsinejad-Kalahroudi, V., Kamalidehghan, B., Arasteh Kani, A., Aryani, O., Tondar, M., Ahmadipour, F., Chung, L.Y. and Houshmand, M., 2014. Two novel tyrosinase (TYR) gene mutations with pathogenic impact on oculocutaneous albinism type 1 (OCA1). PLoS One, 9: e106656. https://doi.org/10.1371/journal.pone.0106656

Grønskov, K., Dooley, C.M., Østergaard, E., Kelsh, R.N., Hansen, L., Levesque, M.P., Vilhelmsen, K., Møllgård, K., Stemple, D.L. and Rosenberg, T., 2013. Mutations in C10orf11, a melanocyte-differentiation gene, cause autosomal-recessive albinism. Am. J. Hum. Genet., 92: 415–421. https://doi.org/10.1016/j.ajhg.2013.01.006

Gul, H., Ali, M.Z., Khan, E., Zubair, M., Badar, M., Khan, S., Shah, A.H. and Khan, M.A., 2017. Ophthalmo-genetic analysis of Pakistani patients with nonsyndromic oculocutaneous albinism through whole exome sequencing. J. Pak. med. Assoc., 67: 790–792.

Gul, H., Shah, A.H., Harripaul, R., Mikhailov, A., Prajapati, K., Khan, E., Ullah, F., Zubair, M., Ali, M.Z., Shah, A.H., Salman, S., Khan, S., Vincent, J.B. and Khan, M.A., 2019. Genetic studies of multiple consanguineous Pakistani families segregating oculocutaneous albinism identified novel and reported mutations. Annls Hum. Genet., 83: 278–284. https://doi.org/10.1111/ahg.12307

Hamid, M.A., Mehta, M.C. and Kuppermann, B.D., 2018. Multimodal imaging in a patient with Prader–Willi syndrome. Int. J. Retina Vitreous, 4: 45. https://doi.org/10.1186/s40942-018-0147-6

Hutton, S.M. and Spritz, R.A., 2008. Comprehensive analysis of oculocutaneous albinism among non-hispanic caucasians shows that OCA1 is the most prevalent OCA type. J. Invest. Dermatol., 128: 2442–2450. https://doi.org/10.1038/jid.2008.109

Inagaki, K., Suzuki, T., Ito, S., Suzuki, N., Adachi, K., Okuyama, T., Nakata, Y., Shimizu, H., Matsuura, H., Oono, T., Iwamatsu, H., Kono, M. and Tomita, Y., 2006. Oculocutaneous albinism type 4: Six novel mutations in the membrane-associated transporter protein gene and their phenotypes. Pigment Cell Res., 19: 451–453. https://doi.org/10.1111/j.1600-0749.2006.00332.x

Jaworek, T.J., Kausar, T., Bell, S.M., Tariq, N., Maqsood, M.I., Sohail, A., Ali, M., Iqbal, F., Rasool, S., Riazuddin, S., Shaikh, R.S. and Ahmed, Z.M., 2012. Molecular genetic studies and delineation of the oculocutaneous albinism phenotype in the Pakistani population. Orphanet J. Rare Dis., 7: 44. https://doi.org/10.1186/1750-1172-7-44

Kamaraj, B. and Purohit, R., 2014. Mutational analysis of oculocutaneous albinism: A compact review. BioMed Res. Int., 2014: 1–10. https://doi.org/10.1155/2014/905472

Kausar, T., Bhatti, M., Ali, M., Shaikh, R. and Ahmed, Z., 2013. OCA5, a novel locus for non-syndromic oculocutaneous albinism, maps to chromosome 4q24. Clin. Genet., 84: 91–93.

Konno, T., Abe, Y., Kawaguchi, M., Storm, K., Biervliet, M., Courtens, W., Kono, M., Tomita, Y. and Suzuki, T., 2009. Oculocutaneous albinism type IV: A boy of Moroccan descent with a novel mutation in SLC45A2. Am. J. Med. Genet. A, 149A: 1773–1776. https://doi.org/10.1002/ajmg.a.32964

Kozakov, D., Hall, D.R., Xia, B., Porter, K.A., Padhorny, D., Yueh, C., Beglov, D. and Vajda, S., 2017. The ClusPro web server for protein–protein docking. Nat. Protoc., 12(2): 255–278. https://doi.org/10.1038/nprot.2016.169

Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J. and Higgins, D.G., 2007. Clustal W and Clustal X version 2.0. Bioinformatics, 23: 2947–2948. https://doi.org/10.1093/bioinformatics/btm404

Morice-Picard, F., Lasseaux, E., François, S., Simon, D., Rooryck, C., Bieth, E., Colin, E., Bonneau, D., Journel, H., Walraedt, S., Leroy, B.P., Meire, F., Lacombe, D. and Arveiler, B., 2014. SLC24A5 mutations are associated with non-syndromic oculocutaneous albinism. J. Investigat. Dermatol., 134: 568–571. https://doi.org/10.1038/jid.2013.360

Muzammal, M., Ali, M.Z., Brugger, B., Blatterer, J., Ahmad, S., Taj, S., Shah, S.K., Khan, S., Enzinger, C., Petek, E., Wagner, K., Khan, M.A. and Windpassinger, C., 2022. A novel protein truncating mutation in L2HGDH causes L-2-hydroxyglutaric aciduria in a consanguineous Pakistani family. Metab. Brain Dis., 37: 243–252. https://doi.org/10.1007/s11011-021-00832-2

Norman, C.S., O’Gorman, L., Gibson, J., Pengelly, R.J., Baralle, D., Ratnayaka, J.A., Griffiths, H., Rose-Zerilli, M., Ranger, M., Bunyan, D., Lee, H., Page, R., Newall, T., Shawkat, F., Mattocks, C., Ward, D., Ennis, S. and Self, J.E., 2017. Identification of a functionally significant tri-allelic genotype in the Tyrosinase gene (TYR) causing hypomorphic oculocutaneous albinism (OCA1B). Sci. Rep., 7: 4415. https://doi.org/10.1038/s41598-017-04401-5

Okamura, K., Hayashi, M., Abe, Y., Kono, M., Nakajima, K., Aoyama, Y., Nishigori, C., Ishimoto, H., Ishimatsu, Y., Nakajima, M., Hozumi, Y. and Suzuki, T., 2019. NGS-based targeted resequencing identified rare subtypes of albinism: Providing accurate molecular diagnosis for Japanese patients with albinism. Pigment Cell Melanoma Res., 32: 848–853. https://doi.org/10.1111/pcmr.12800

Passmore, L., Kaesmann-Kellner, B. and Weber, B., 1999. Novel and recurrent mutations in the tyrosinase gene and the P gene in the German albino population. Hum. Genet., 105: 200-210. https://doi.org/10.1007/s004399900104

Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C. and Ferrin, T.E., 2004. UCSF Chimera a visualization system for exploratory research and analysis. J. Comput. Chem., 25: 1605–1612. https://doi.org/10.1002/jcc.20084

Preising, M.N., Forster, H., Tan, H., Lorenz, B., de Jong, P.T.V.M. and Plomp, A.S., 2007. Mutation analysis in a family with oculocutaneous albinism manifesting in the same generation of three branches. Mol. Vis., 13: 1851-1855. http://www.molvis.org/molvis/v13/a206/

Sengupta, M., Mondal, M., Jaiswal, P., Sinha, S., Chaki, M., Samanta, S. and Ray, K., 2010. Comprehensive analysis of the molecular basis of oculocutaneous albinism in Indian patients lacking a mutation in the tyrosinase gene. Br. J. Dermatol., 163: 487–494. https://doi.org/10.1111/j.1365-2133.2010.09830.x

Shah, S.A., Din, S.U., Raheem, N., Daud, S., Mubeen, J., Nadeem, A., Tayyab, M., Baloch, D.M., Babar, M.E., and Ahmad, J., 2014. Identification of a novel mutation (p.Ile198Thr) in gene TYR in a Pakistani family with nonsyndromic oculocutaneous albinism. Clin. Exp. Dermatol., 39: 646–648. https://doi.org/10.1111/ced.12382

Shahzad, M., Yousaf, S., Waryah, Y.M., Gul, H., Kausar, T., Tariq, N., Mahmood, U., Ali, M., Khan, M.A., Waryah, A.M., Shaikh, R.S., Riazuddin, S., Ahmed, Z.M., Bamshad, M.J., Shendure, J., Nickerson, D.A., Abecasis, G.R., Anderson, P., Marchani Blue, E. and Yi, Q., 2017. Molecular outcomes, clinical consequences, and genetic diagnosis of oculocutaneous albinism in Pakistani population. Sci. Rep., 7: 44185.

Shakil, M., Harlalka, G.V., Ali, S., Lin, S., D’Atri, I., Hussain, S., Nasir, A., Shahzad, M.A., Ullah, M.I., Self, J.E., Baple, E.L., Crosby, A.H. and Mahmood, S., 2019. Tyrosinase (TYR) gene sequencing and literature review reveals recurrent mutations and multiple population founder gene mutations as causative of oculocutaneous albinism (OCA) in Pakistani families. Eye, 33: 1339–1346. https://doi.org/10.1038/s41433-019-0436-9

Sievers, F. and Higgins, D.G., 2014. Clustal Omega. Curr. Protoc. Bioinf., 48. https://doi.org/10.1002/0471250953.bi0313s48

Simeonov, D.R., Wang, X., Wang, C., Sergeev, Y., Dolinska, M., Bower, M., Fischer, R., Winer, D., Dubrovsky, G., Balog, J.Z., Huizing, M., Hart, R., Zein, W.M., Gahl, W.A., Brooks, B.P. and Adams, D.R., 2013. DNA Variations in oculocutaneous albinism: An updated mutation list and current outstanding issues in molecular diagnostics. Hum. Mutat., 34: 827–835. https://doi.org/10.1002/humu.22315

Summers, C.G., 2009. Albinism: Classification, clinical characteristics, and recent findings. Optomet. Vision Sci., 86: 659–662. https://doi.org/10.1097/OPX.0b013e3181a5254c

Szklarczyk, D., Gable, A.L., Lyon, D., Junge, A., Wyder, S., Huerta-Cepas, J., Simonovic, M., Doncheva, N.T., Morris, J.H., Bork, P., Jensen, L.J. and Mering, C., 2019. STRING v11: Protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucl. Acids Res., 47: D607–D613. https://doi.org/10.1093/nar/gky1131

Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B.C., Remm, M. and Rozen, S.G., 2012. Primer 3 New capabilities and interfaces. Nucl. Acids Res., 40: e115–e115. https://doi.org/10.1093/nar/gks596

Yang, J., Yan, R., Roy, A., Xu, D., Poisson, J. and Zhang, Y., 2015. The I-TASSER Suite: Protein structure and function prediction. Nat. Methods, 12: 7–8. https://doi.org/10.1038/nmeth.3213

Zhang, Y., Zhang, Y., Liu, T., Bai, D., Yang, X., Li, W. and Wei, A., 2019. Identification of two Chinese oculocutaneous albinism type 6 patients and mutation updates of the SLC 24A5 gene. J. Dermatol., 46: 1027–1030. https://doi.org/10.1111/1346-8138.15065