Identification of Pathogenic Mutation c.286dupA in TYR Gene in an Individual with Oculocutaneous Albinism Using Exome Sequencing

Objective To investigate gene mutations in a pedigree with oculocutaneous albinism by using targeted next-generation sequencing technology. Methods Clinical data were collected from a pedigree with oculocutaneous albinism. Genomic DNA was extracted from peripheral blood cells of the proband and his parents. High-throughput sequencing technology was used for sequence analysis of coding regions in exons of 29 genes including TYR, OCA2, TYRP1 and SLC45A2 in the proband to find potential pathogenic gene mutations. Sanger sequencing was conducted to detect the corresponding genetic loci in the parents. Results Two heterozygous mutations were identified in the TYR gene of the proband, including a novel mutation c.534G > C (p.Trp178Cys) and a known mutation c.1147G > A (p.Asp383Asn) . The detection of the TYR gene mutations in the parents of the proband showed that the c.534G > C and c.1147G > A mutations in the proband were inherited from his father and mother respectively. Conclusion A novel pathogenic mutation c.534G > C in the TYR gene is identified in the pedigree with oculocutaneous albinism by using targeted next-generation sequencing technology. Key words: Albinism, oculocutaneous; Genotyping techniques; Mutation; Prenatal diagnosis

Oculocutaneous albinism is a rare genetic disorder characterized by the absence or reduction of melanin pigment in the skin, hair, and eyes, leading to various visual and dermatological challenges. To shed light on the molecular pathology of OCA in consanguineous Pakistani families, we conducted whole-exome sequencing on affected individuals from two families. We identified disease-causing homozygous mutations in the TYR (NM_000372) and OCA2 (NM_000275.3) genes that segregated within their respective pedigrees. In family AL01, we identified a novel mutation in the TYR gene, resulting in a missense change, c.1280T>C, leading to p.V427A. In family AL02, we detected a splice site variant, c.1045-15T>G in OCA2 gene. Protein model of the V427A mutation within the tyrosinase protein predicted that as the mutant amino acid was considerably smaller in size than the wild type, it might have created a potential gap in protein's core structure. The V427A mutation is positioned centrally within the Lumenal melanosome repeat domain raises concerns about potential structural alterations in this domain due to disparities between the wild-type and mutant residue, potentially leading to a loss of function in this repeated region. Our study provides a deeper understanding of the molecular basis of OCA in consanguineous Pakistani families by identifying disease-causing mutations in the TYR and OCA2 genes. The novel TYR mutation, V427A, offers insights on the structural consequences of this mutation, which could have implications for understanding the pathology of OCA and potentially effecting future diagnostic and therapeutic approaches for individuals affected by this rare genetic disorder.

BackgroundOculocutaneous albinism (OCA) is an autosomal recessive disorder. The most common type OCA1 and OCA2 are caused by homozygous or compound heterozygous mutations in the tyrosinase gene (TYR) and OCA2 gene, respectively.ObjectiveThe purpose of this study was to evaluate the molecular basis of oculocutaneous albinism in four Chinese families.Patients and MethodsFour non-consanguineous OCA families were included in the study. The TYR and OCA2 genes of all individuals were amplified by polymerase chain reaction (PCR), sequenced and compared with a reference database.ResultsFour patients with a diagnosis of oculocutaneous albinism, presented with milky skin, white or light brown hair and nystagmus. Genetic analyses demonstrated that patient A was compound heterozygous for c.1037-7T.A, c.1037-10_11delTT and c.1114delG mutations in the TYR gene; patient B was heterozygous for c.593C>T and c.1426A>G mutations in the OCA2 gene, patients C and D were compound heterozygous mutations in the TYR gene (c.549_550delGT and c.896G>A, c.832C>T and c.985T>C, respectively). The heterozygous c.549_550delGT and c.1114delG alleles in the TYR gene were two novel mutations. Interestingly, heterozygous members in these pedigrees who carried c.1114delG mutations in the TYR gene or c.1426A>G mutations in the OCA2 gene presented with blond or brown hair and pale skin, but no ocular disorders when they were born; the skin of these patients accumulated pigment over time and with sun exposure.ConclusionThis study expands the mutation spectrum of oculocutaneous albinism. It is the first time, to the best of our knowledge, to report that c.549_550delGT and c.1114delG mutations in the TYR gene were associated with OCA. The two mutations (c.1114delG in the TYR gene and c.1426A>G in the OCA2 gene) may be responsible for partial clinical manifestations of OCA.

Dear Sir, Oculocutaneous albinism (OCA) is an autosomal recessive disease of skin, hair and eye hypopigmentation caused by a deficiency in melanin biosynthesis. Four types of non-syndromic OCA are described. OCA1 (MIM203100), with OCA1A and OCA1B subtypes, is caused by mutations in the TYR gene that spans 118 kb in 11q14.3 and contains five exons. About 200 mutations of TYR have been described (Albinism database, http://albinismdb.med.umn.edu/). OCA2 (MIM203200) is caused by mutations in the OCA2 gene that spans 345 kb in 15q11.2q12 and contains 25 exons. To date, about 80 mutations have been described in the OCA2 gene (Albinism Database). OCA3 (MIM203290) is caused by mutations in TYRP1 leading to rufous OCA. The TYRP1 gene spans 17 kb in 9p23 and is composed of eight exons. Only five mutations were described so far in TYRP1 (Albinism Database). OCA4 (MIM606574) is caused by mutations in SLC45A2 that spans 40 kb in 5p13.3 with seven exons. To date, 30 mutations have been identified in Turkish, German, Korean, Japanese, Brazilian, and Indian patients (Albinism Database). In this paper, we analyze a series of 63 patients with OCA and describe novel mutations (including intragenic deletions) in the four OCA genes and propose a molecular analysis strategy based on the re-evaluation of frequency of the different OCA types. Genotype/phenotype correlations are discussed. The search for point mutations was performed either by DHPLC analysis followed by sequencing of the variants or by direct sequencing, depending on the genes and exons. Intragenic deletions were screened by quantitative multiplex fluorescent PCR (QMF PCR) (Niel et al., 2004). Primer sequences and experimental conditions are available from the authors upon request. Twenty nine OCA patients were found to carry mutations in TYR (46%; OCA1). We identified 31 different mutations including 13 novel ones: three splice site mutations, three nonsense mutation, three missense mutations, one frameshift, one deletion of three nucleotides, one deletion of the entire gene, and one deletion of a single exon (Table 1). Twenty-three patients harbored two mutations (79%). Six patients carried only one TYR mutation (21%), the second one remaining uncovered yet. The potential 5′ distal regulatory sequences including the Terminal Distal Element (TDE), the Locus Control Region (LCR) and a large complex GA repeat were analyzed in these patients (Ray et al., 2007). No mutation was found in the LCR or TDE, and no significant difference in the GA repeat size was observed between the six patients analyzed (size range: 297–317 bp) and 47 controls (size range: 307–317 bp). Our data therefore suggest that a variation in the length of this promoter polymorphism is not involved in OCA pathogenesis. A deletion of the entire TYR gene was found in a patient (97066) presenting with a typical OCA1A form and leukodystrophy (Coupry et al., 2001; Goizet et al., 2004). A new heterozygous deletion of a large part of exon 2 was identified in patient 070996. Two patients had such a mild skin involvement that a pure ocular albinism could be discussed (patient 070965 in Table 1 and patient 060611 with two heterozygous known mutations: p.His367Tyr and p.Arg402Gln). Patient 060058 from consanguineous Moroccan parents, with positive tyrosinase testing on hair bulb, was homozygous for a new splice site mutation (c.1185-1 G > T). This variant might represent a new OCA1B mutation. On the contrary, patient 050813 had a severe phenotype, although he carried one OCA1B mutation (p.Val275Phe) and a novel probably highly pathogenic missense mutation (p.His19Arg) occurring next to the signal peptide, early in the protein. OCA2 mutations were found in 18 patients (29%). Seventeen patients carried two OCA2 mutations or deletions while one patient carried only one mutation. We identified 13 novel mutations including seven missense, two frameshift, one splicing, one nonsense mutation, and two deletions spanning multiple exons (Table 1). Most of the missense mutations occur in the loops between the transmembrane domains as already described (Spritz et al., 1997). The recurrent 'African' deletion including exon 7 was found in six patients. Four patients were from Africa (Angola, Cameroon, Guinea, Zaire), one from Guadeloupe (with African ancestors), and one from Spain (without information about ancestors). All had an OCA2 phenotype with light skin, yellow to light brown hair, and blue to light brown eyes. Several new intragenic deletions were detected in OCA2. Two patients (030857, 070606) carried intragenic deletions including exons 3–20. In the current state of analysis, we cannot assure that the two deletions are formally identical. Patient 070606 was homozygous for the deletion, although no consanguinity was known in her family. She had blond hair and light skin, compatible with an OCA2 skin phenotype, with a quite severe ocular phenotype. It is notable that several deletions of the 15q11.2 region have been reported. Altogether, these data indicate the frequent occurrence of recombination mechanisms in this gene. In a Turkish patient (040578) with OCA and a clinical phenotype of Angelman Syndrome, we identified a heterozygous OCA2 missense mutation (p.Pro198Leu), and a 6 Mb deletion comprising the OCA2 and UBE3A genes (using 15q11.2q12 microsatellites, QMF-PCR, and array-CGH 44K from Agilent Technologies; data not shown) The breakpoints were refined to regions of 600 and 800 kb in the centromeric 'BP2', and in the telomeric 'BP3' hotspots, respectively (Christian et al., 1999). One patient (030791) may represent a case of digenism. He carried known missense mutations in TYR (p.Thr373Lys) and OCA2 (p.Arg290Gly), both in the heterozygous state. His mother did not carry any mutation whereas his father carried the two mutations. We therefore assume that these two events were not sufficient to lead to the OCA phenotype in this patient and that a third event has occurred and needs to be identified. No other mutation was found so far in the explored regions of each of the four OCA genes in this patient. Two patients were found to be mutated in TYRP1 (3%) (OCA3), and the four mutations identified were novel ones (Table 1). Patient 02513 was already described by us (Rooryck et al., 2006). Patient 020513 harbored two heterozygous mutations in TYRP1 and one splicing mutation in TYR that abolishes the donor site of intron 4. Her mother carried the TYRP1 mutation in exon 5, and her father carried the TYRP1 mutation in exon 4 as well as the TYR mutation. Both parents were healthy, thus excluding the possibility of a digenism, but not of a triallelism. This patient had a quite classical OCA3 phenotype. Finally, one African patient (050372) had two mutations in OCA2 and one nonsense variant in TYRP1, thus raising the possibility of a triallelism. This variant has been described as a polymorphism (rs41302073, NCBI), which is doubtful for a nonsense mutation. The patient's mother carried only one OCA2 mutation and the father could not be analyzed. Further investigation of these two cases of possible triallelism would require testing more family members. Eleven patients were identified with mutations in the SLC45A2 gene (17%) (OCA4). Three patients had a known recurrent mutation (c.986delC) in the homozygous state and five patients carried new mutations (Table 1). The c.986delC mutation is not restricted to the German population as suggested before (Rundshagen et al., 2004) as we found it in two Spanish and one French patients. Patient 050588 carried a novel missense mutation and a deletion of exon 4. This is the first SLC45A2 intragenic deletion to be reported. The patient's phenotype was quite severe at birth but became milder with age. Finally, patient 030878 carried a missense mutation, and a new variant in the promoter of SLC45A2, a region that was functionally characterized by (Graf et al., 2007). This variant was found in the heterozygous state in two of 100 controls. Functional analyses on RNA extracted from skin melanocytes or hair bulbs are needed in order to confirm the role of this variant. The phenotype in our OCA4 patients is variable and seems to depend upon the mutation type: it ranges from severe (nonsense or frameshift mutations) to milder forms of OCA (missense mutations, mutations in the promoter). Practically, the strategy for molecular analysis of OCA patients can be partially adapted according to the clinical form of the OCA, to tyrosinase activity measurements (on hair bulb or skin biopsy), and to the patient's ethnicity. Tyrosinase-negative patients will be tested for TYR first, whereas tyrosinase-positive patients will have OCA2 tested first. In our experience, the tyrosinase activity test on hair bulb proved to be robust only if performed in patients older than 2 yr and if the hair bulb was analyzed within 72 h from sampling (unpublished data). Quite characteristically, one of our patients (061001) with OCA4 was tyrosinase-negative at the age of 1 yr, but turned tyrosinase-positive when she was 3 yr old. The patient's ethnic origin comes also into consideration in defining the investigatory strategy, since the different forms of OCA are not represented equally in the various populations worldwide. Since OCA1 is more prevalent than OCA2 in Europe, a Caucasian patient from Europe will therefore have the TYR gene tested first. On the other hand, a Black African patient will have OCA2 screened in a first instance, with the prevalent exon 7 deletion being searched for first, and point mutations and other deletions being searched for next. OCA1A and B subtypes correspond to different TYR mutations. The nonsense, frameshift, and the missense mutations that completely abolish the tyrosinase catalytic activity (probably including the novel one p.His19Arg) cause OCA1A, whereas missense mutations such as p.Val275Phe, p.Pro406Leu, p.Arg422Gln, and the novel splicing mutation c.1185-1 G > T lead to OCA1B. The p.Arg402Gln variant is classically considered as a polymorphism though it has been proved by several authors that it causes the thermo-sensitive retention of TYR in the endoplasmic reticulum of HeLa cells and melanocytes (Berson et al., 2000; Halaban et al., 2000). We found p.Arg402Gln in the heterozygous state in association with another TYR mutation in trans in several OCA patients, and we therefore suggest that this variant should now be considered as a genuine mild mutation. Depending upon the type of mutation it is associated with, p.Arg402Gln can be found in patients with either OCA1A or OCA1B. SLC45A2 mutations were found to be more prevalent (17%) in our data set than previously described, i.e. 5–6% of OCA in Caucasians (Rundshagen et al., 2004), and 10% in Indians (Sengupta et al., 2007). Other authors reported that only one mutation was identified in one-third of patients (Sengupta et al., 2007), while we identified both mutations in our five OCA4 patient. SLC45A2 should therefore be tested before TYRP1, which represents the least frequent form of OCA worldwide. To conclude, we have analyzed 63 patients and described 37 novel mutations in the four OCA genes. Two mutations were identified in 52 patients of the 63 from our series (83%). The thorough analysis of the four genes, including the search for intragenic rearrangements, as well as, when available, the analysis of the promoter regions, proved important in order to attain this high yield of mutation identification. It should be noted that 10% of all mutated alleles were deletions. A single mutation was identified in seven cases (six patients with a TYR mutation and one with an OCA2 mutation), thus indicating that mutations remained uncovered either in one of the four OCA1–4 genes or in an as yet unidentified gene(s). In these patients and in those in whom no mutation had been identified, we explored the DCT gene (dopachrome tautomerase), coding for TYRP2, a possible OCA candidate gene. No mutation was found in the 10 exons of the gene. Although our series of patients is not representative of the worldwide population, it provides a comprehensive study of OCA patients from different origins (mainly Europeans) and allowed us to estimate the relative frequencies of the different forms of OCA: 46% OCA1, 29% OCA2, 3% OCA3, and 17% OCA4. Five percent of cases remain unresolved. Our data highlight the fact that OCA classification best relies on molecular analysis of the four genes as almost identical phenotypes can be observed in OCA1–4. The authors thank the french Ministry of Research of France, the french Ministry of Health, and the Association Genespoir for their financial support. Part of the experimental work was performed on the Genotyping and Sequencing Facility of Bordeaux which was established thanks to grants from the Conseil Régional d'Aquitaine (no. 20030304002FA and no. 20040305003FA) and from the FEDER (no. 2003227).

Oculocutaneous albinism (OCA) is a group of heterogeneous and autosomal recessive disorders characterized by a reduction or complete loss of melanin biosynthesis in melanocytes. OCA type1 (OCA1) is the most severe and common form of OCA, and is caused by mutations in the tyrosinase gene (TYR). The present study aimed to identify the genetic cause of OCA1 in a four‑generation consanguineous Chinese Han family. Complete physical examinations were performed and blood samples were collected from five members of the family and 100unrelated healthy controls. Exome sequencing was conducted in the proband, followed by verification in other family members, using Sanger sequencing. Patients in the family presented with typical OCA1 features, including hypopigmentation of the skin and hair, and distinctive ocular changes. A homozygous missense variant, c.896G>A (p.R299H), in the TYR gene was identified in two patients, which co‑segregated with disease in the family. This variant was not present in the 100healthy controls. These results expand the number of mutations identified to be responsible for OCA1 in the Chinese Han population, and may have implications for genetic counseling and clinical management of the disease.

Oculocutaneous albinism (OCA) is an autosomal recessive pigmentation abnormality, characterized by variable hair, skin, and ocular hypopigmentation. OCA1 is the most frequent subtype of OCA, caused by mutations in the tyrosinase gene (TYR). In this study, we investigated the genetic mutation of a Chinese family with a female OCA patient who came for genetic counseling before pregnancy. Complete physical examination was performed, and DNA from blood samples was collected from the family members. Mutations of TYR, OCA2, and SLC45A2 genes were examined in the proband, and verified in her parents by Sanger sequencing. Large deletion or duplication of TYR and OCA2 genes was detected by multiplex ligation-dependent probe amplification (MLPA). A homozygous TYR c.307T>C (p.Cys103Arg) missense mutation was identified in the proband, and both parents were heterozygous carriers. No large deletion or duplication was found in the proband. This mutation was absent in 1000G, ExAC, or HGMD database, and multiple lines of in silico tools supported a deleterious effect. These results suggest that TYR c.307T>C mutation might be responsible for OCA1, and our study further expands the mutation spectrum of OCA1 in the Chinese population.

Melanoma, the deadliest type of skin cancer, derives from the transformation of melanocytes, which protect the skin from ultraviolet radiation (UVR) through the synthesis of melanin. UVR is the major risk factor for melanoma. Intriguingly, in people with oculocutaneous albinism (OCA), who lack or have a reduction in melanin synthesis melanoma is surprisingly rare, whereas other UVR-driven skin cancers are comparatively high. Since UVR exposure and light skin complexion increase melanoma risk, it is unclear why melanoma is rare in OCA. Therefore, we evaluated how the absence of melanin impact the mutation burden in OCA and identify driver mutations by whole exome sequencing (WES) analysis of melanomas from a patient with OCA. The patient (in their 50s) presented cutaneous basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) in addition to melanoma. The histopathology of one tumor (sample A), located on the left forearm, indicated a desmoplastic nodular melanoma, Clark level III, Breslow of 1 mm and a high mitotic index. The second tumor (sample B) was located on the right knee and presented a superficial spreading melanoma, Clark level II, a Breslow of 0.31 mm and a low mitotic index. For WES, genomic DNA was purified from formalin-fixed paraffin-embedded tissue sections and compared against germline blood. Analysis confirmed a G47D mutation in the TYR gene, which has previously been reported in OCA1B patients. Consistent with sample A coming from an area habitually exposed to UVR, it presented 4874 SNVs and 1556 missense SNVs and a predominant signature 7 mutational process. Similarly, consistent with sample B coming from an area that is habitually protected, it presented only 45 SNVs and 16 missense SNVs and did not present a predominant signature 7 mutational process. Notably, despite the histopathology of the skin adjacent to sample B appeared normal, it presented 39 SNVs and 10 missense SNVs. Intriguingly, all samples harbor NRASQ61K mutations and sample B additionally presented a mutation in NOTCH1, which is more common in SCC. Thus, we show that in OCA, melanomas from a UVR exposed areas present high mutation burden and UV mutational signature, whereas melanomas from UVR protected skin present a low mutation burden. Our data suggest that NRASQ61K is a driver oncogene in melanomas from albinos and that these patients can develop both UVR-driven and UVR-independent melanomas. Citation Format: Candelaria Bracalente, Piyushkumar Mundra, Adriana Rinflerch, Pablo Garcia Martinez, Victoria Volonteri, Lucas Trucco, Gaston Galimberti, Nathalie Dhomen, Valeria Pavet Rodriguez, Richard Marais. Genetic analysis of melanoma from an albino patient [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3438.

Unveiling genetics of non-syndromic albinism using whole exome sequencing: A comprehensive study of TYR, TYRP1, OCA2 and MC1R genes in 17 families

Next-generation sequencing (NGS) of the whole genome, whole exome, and transcriptome has enabled characterization of genetic landscapes of multiple cancers. By analyzing over 2,000 pediatric cancer patients, we have developed a comprehensive database for recurrent somatic alterations and pathogenic germline mutations as part of the St. Jude/Washington University Pediatric Cancer Genome Project. However, there is no systematic evaluation on whether NGS is able to identify germline and somatic lesions reported by existing molecular diagnostic assays and what combination of NGS platforms is best suited for clinical sequencing. Here we report the first comprehensive study that employs whole-genome sequencing at 30-45X coverage, whole-exome sequencing at 100X coverage and transcriptome sequencing using matched tumor/normal samples from cancer patients. A pilot study was carried out to perform NGS analysis on 78 children of leukemia, solid tumor or brain tumor with a total of 112 diagnostic or prognostic biomarkers previously characterized by multiple molecular diagnostic assays. We implemented an analysis pipeline that integrates the genetic lesions detected by all three NGS platforms to characterize somatic and germline single nucleotide variations (SNVs), short insertions and deletions (indels), structural variations including fusions, karyotypes, copy number alterations, loss of heterozygosity, tumor purity and tumor-in-normal contamination. The turn-around time for data analysis is 2 weeks with an overall sensitivity of 99% on detecting known biomarkers. Extensive validation of >3,000 somatic sequence mutations or structural variations from 38 cases shows that the specificity for somatic SNV, indel and structural variation is at 98%, 95% and 84% across the genome. We demonstrate that in addition to providing cross-validation, multi-platform NGS is required for detecting all genetic lesions of pathological significance including complex re-arrangements such as chromothripsis. In addition to known pathogenic or likely pathogenic mutations, our analysis has also unveiled novel pathogenic mutations (e.g. a germline deletion in TP53 in one patient with medulloblastoma) and identified multiple variants of unknown significance that may be worth further exploration (e.g. an in-frame deletion of exons 3-9 of DNMT3A in one neuroblastoma). Our study demonstrates that NGS is able to detect a wide range of genetic lesions currently characterized by multiple molecular diagnostic assays, providing critical insight into the design of clinical sequencing for ongoing studies. Citation Format: Jinghui Zhang, Michael Rusch, Joy Nakitandwe, Zhaojie Zhang, Michael N. Edmonson, Matthew Parker, Xiaotu Ma, Jared Becksfort, Andrew Thrasher, Jiali Gu, Yongjin Li, Erin Hedlund, Aman Patel, John Easton, Donald Yergeau, Bhavin Vadodaria, Xiang Chen, Tanja A. Gruber, Rose McGee, David Ellison, Sheila Shurtleff, James R. Downing. Molecular diagnosis for pediatric cancer through integrative analysis of whole-genome, whole-exome and transcriptome sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2628.

Background: Whole exome sequencing (WES), which focuses on sequencing of protein-coding regions of human genome, has greatly improved the identification of causal mutations for diverse human genetic disorders in the past four years. We applied WES as a molecular diagnostic tool to identify disease-causing mutations in consanguineous families displaying autosomal recessive (AR) disorders in Qatar. AR diseases are usually severe and rare that occurs with higher rate in consanguineous families. In Qatar, like other Middle East countries, consanguineous marriage and endogamy are common (54%) that results in a higher incidence of several/new AR disorders where many of these disorders are yet to be defined and their causative genes are to be discovered. In order to decrease the overall socio-economic burden of such diseases in the society and development of specific testing tools that will be helpful with the molecular diagnosis, parental testing, carrier identification, and informed genetic counseling, we must first discover the causative genes of these recessive disorders. Methods: All of the experimental (whole exome library preparation, capturing and sequencing) and bioinformatics analyses were performed according to our well-established protocols at Genome Quebec Innovation Center, Montreal, Canada. Results: We performed WES on several consanguineous families, with one or more affected children, where the result of initial molecular screening of known or potential candidate genes was negative. Because of unaffected status of parents and their consanguinity, the mode of inheritance was considered to be autosomal recessive. We also tested de novo and X-linked mode of inheritance in families with one or only male affected child. Based on these assumptions, we identified the definitive damaging mutations for Hypophosphatemic rickets; Hurler syndrome; Glycogen storage disease; Dubowitz-like syndrome; Seckel syndrome; Geleophysic dysplasia; Limb-girdle muscular dystrophy; Multiple Fractures; Metachromatic Leukodystrophy; Immunodeficiency and Juvenile onset cataract. In addition, several candidate genes were identified in families with mental retardation (n=3); CNS anomaly (n=1); eye anomalies (n= 26); peripheral neuropathy (n=7); axonal peripheral neuropathy (n=3) and oro-facio-digital syndrome (n=2), that have been considered for functional follow-up investigations and further characterizations. Conclusion: Our study highlights the importance of using WES as molecular diagnostic approach for discovery pathogenic gene mutations compared to traditional molecular genetic testing. We showed that the WES was successful to identify causal mutations underlying phenotypically complex disorders in ~46% of our patients. The results of this study will help to establish population-specific diagnostic panels, and improve clinical diagnosis and patient management in the country.

Oculocutaneous albinism (OCA) is associated with a wide range of clinical presentations and has been categorized with syndromic and non-syndromic features. The most common causative genes in non-syndromic OCA are TYR and OCA2 and HSP1 is in the syndromic albinism. The objective of this study was to identify pathogenic variants in congenital OCA families from Pakistan. Eight consanguineous families were recruited, and clinical and ophthalmological examination was carried out to diagnose the disease. Whole blood was collected from the participating individuals, and genomic DNA was extracted for sequencing analysis. TruSight one-panel sequencing was carried out on one affected individual of each family, and termination Sanger sequencing was carried out to establish the co-segregation of the causative gene or genes. In silico analysis was conducted to predict the causative pathogenic variants. Two families were found to have novel genetic pathogenic variants, and six families harbored previously reported variants. One novel compound heterozygous pathogenic variant in the TYR gene, c.1002delA; p.Ala335LeufsTer20, a novel frameshift deletion pathogenic variant and c.832C>T; and p.Arg278Ter (a known pathogenic variant) were found in one family, whereas HPS1; c.437G>A; and p.Trp146Ter were detected in another family. The identification of new and previous pathogenic variants in TYR, OCA2, and HPS1 genes are causative of congenital OCA, and these findings are expanding the heterogeneity of OCA.

Whole Exome Sequencing Identifies Novel Lyst-Missense Mutations In Incomplete Childhood Chediak-Higashi-Syndrome Presenting As Hemphagocytic Lymphohistiocytosis (HLH)

Oculocutaneous albinism (OCA) is a congenital heterogeneous group of autosomal recessive disorders characterized by the absence or loss of melanin in the skin, eyes and hair of the affected individuals. Based on the mutated gene, OCA has been classified into eight sub-types (OCA1-8) with overlapping clinical phenotypes. Mutations in the TYR gene cause OCA1, the most prevalent OCA worldwide including India. Mutations in OCA2 and SLC45A2, both of which regulate melanosomal pH that is critical to TYR activity, cause OCA2 and OCA4 respectively, the other common OCA subtypes in India. In the present study, we have included 54 OCA-affected cases from 41 unrelated families representing 16 different marriage/ethnic groups from 17 districts of West Bengal, India. We pursued a PCR-sequencing based approach followed by bioinformatic analysis to identify mutations in TYR, OCA2 and SLC45A2 genes. Mutations were detected in 27 of the 54 (50%) OCA patients from 18 unrelated families, representing 9 different marriage/ethnic groups from 11 districts of West Bengal. Three TYR variants: NM_000372.4: c.391 A > G, NP_000363.1: p. Lys131Glu; NM_000372.4: c.1037G > T; NP_000363.1: p. Gly346Val, NM_000372.4: c.715 C > T; NP_000363.1:p.Arg239Trp was identified for the first time in Eastern Indian OCA cases. A novel nonsense variant: NM_016180.5: c.389T > A, NP_057264.4: p. Leu130* and a novel synonymous variation NM_016180.5: c.1092 A > G; NP_057264.4: p.364E = were identified in SLC45A2. Additionally, NM_016180.5: c.904A > T; NP_057264.4: p. Thre302Ser was identified for the first time in any Eastern Indian OCA case. We identified 2 previously reported mutations in OCA2. In concordance with previous reports, NM_000372.4: c.832C > T, NP_000363.1: p. (Arg278*) was the commonest TYR mutation. The results of our study enrich the mutational spectrum of the known OCA causing genes in Eastern India, which would facilitate accurate diagnosis, familial screening, carrier detection and containment of the disease load.

Rapid advances in DNA sequencing technologies have made it increasingly cost-effective to obtain accurate and timely large-scale genomic sequence data on individuals (short read massively parallel or next generation [next-gen]). A next-gen molecular diagnostic approach that has seen rapid deployment in the clinic over the last year is exome sequencing. Whole exome sequencing covers all protein-coding genes in the genome (≈1.1% of genome), and an exome test for a single patient generates ≈6 gigabases (109 bp) of DNA sequence data. A key challenge facing routine use of next-gen data in patient diagnosis and management is data interpretation. What sequence variant findings are relevant to diagnosis (pathogenic mutations)? What sequence variant findings are relevant to clinical care but not necessarily to patient diagnosis (clinically actionable incidental data)? What sequence information should be stored, and where can it be stored? This review provides a tutorial on current approaches to answering these questions. A recent landmark study showed that application of next-gen sequencing to a large cohort of idiopathic dilated cardiomyopathy patients found ≈27% of patients to show mutations of the titin gene, the most complex gene in the genome (363 exons). We use titin in cardiomyopathy as an exemplar for explaining next-gen sequencing approaches and data interpretation. Decreasing sequencing costs and broad dissemination of next-generation (next-gen) equipment and expertise are increasing availability of massively parallel sequencing of patient DNA samples (short read massively parallel or next-gen sequencing).1,2 Most rapidly expanding is exome sequencing, where all protein-coding sequences (exons) are selected from total genomic DNA and selectively sequenced.3 Alternative approaches to next-gen sequencing include targeted sequencing (TS) and whole genome (complete genome) sequencing. Currently, marketed targeted Sanger sequencing panels using traditional individual exon-by-exon sequencing remain expensive and time consuming, and massively parallel next-gen approaches are beginning to supplant …

BackgroundOculocutaneous albinism (OCA) is a group of rare autosomal recessive disorders characterized by clinical genetic heterogeneity. OCA type II (OMIM: 203200) is the most common subtype among African and African Americans, primarily caused by pathogenic variants in the OCA2 (HGNC ID: 8101) gene. In this study, we presented a Chinese family with OCA and reported two novel variants in the OCA2 gene.MethodsWhole‐exome sequencing (WES) was performed to identify pathogenic variants in the proband. The candidate variants were subsequently validated using Sanger sequencing and QPCR assay. Additionally, bioinformatics analyses were employed to predict the deleteriousness and conservation of the identified mutations.ResultsIn the 16‐year‐old male proband, two novel compound heterozygous OCA2 variants, NM_000275.3: c.1640T>G (NP_000266.2: p.L547R) and an exons 10‐19 deletion variant, were identified. Meanwhile, a reported heterozygous variant c.1441G>A/p.A481T (NM_000275.3, NP_000266.2) in the OCA2 gene was also found in the proband. Sanger sequencing confirmed that the two variants c.1441G>A/p.A481T and c.1640T>G/p.L547R were inherited from his father. Moreover, qPCR assay revealed that the exons 10‐19 deletion was inherited from the mother, his sister also carried this variant. Fortunately, the variant was not detected in the amniotic fluid of the proband's sister. Multiple online bioinformatics tools predicted the variant c.1640T>G to be damaging, leading to the replacement of a highly conserved leucine with an arginine. The gross exon 10‐19 deletion in the OCA2 gene resulted in a truncated, non‐functional protein losing the 3–9 transmembrane α‐helices domains. According to the American College of Medical Genetics and Genomics classification, these three variants in the OCA2 gene were evaluated as likely pathogenic.ConclusionThis study has identified two novel compound variants in the OCA2 gene and a previously reported variant in a Chinese family with OCA. By expanding the mutation spectrum of the OCA2 gene, our findings contribute to a better understanding of the genetic basis of OCA.