Germline variants in oculocutaneous albinism genes and predisposition to familial cutaneous melanoma

Approximately 1%–2% of cutaneous melanoma (CM) is classified as strongly familial. We sought to investigate unexplained CM predisposition in families negative for the known susceptibility genes using next‐generation sequencing of affected individuals. Segregation of germline variants of interest within families was assessed by Sanger sequencing. Several heterozygous variants in oculocutaneous albinism (OCA) genes: TYR, OCA2, TYRP1 and SLC45A2, were present in our CM cohort. OCA is a group of autosomal recessive genetic disorders, resulting in pigmentation defects of the eyes, hair and skin. Missense variants classified as pathogenic for OCA were present in multiple families and some fully segregated with CM. The functionally compromised TYR p.T373K variant was present in three unrelated families. In OCA2, known pathogenic variants: p.V443I and p.N489D, were present in three families and one family, respectively. We identified a likely pathogenic SLC45A2 frameshift variant that fully segregated with CM in a family of four cases. Another four‐case family harboured cosegregating variants (p.A24T and p.R153C) of uncertain functional significance in TYRP1. We conclude that rare, heterozygous variants in OCA genes confer moderate risk for CM.     

Mutational Analysis of the TYR and OCA2 Genes in Four Chinese Families with Oculocutaneous Albinism

Background

Oculocutaneous 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.

Objective

The purpose of this study was to evaluate the molecular basis of oculocutaneous albinism in four Chinese families.

Patients and Methods

Four 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.

Results

Four 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.

Conclusion

This 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.     

Oculocutaneous albinism type 4: six novel mutations in the membrane‐associated transporter protein gene and their phenotypes

Oculocutaneous albinism type 4 (OCA4) is an autosomal recessive hypopigmentary disorder caused by mutations in the Membrane‐Associated Transporter Protein gene (SLC45A2). The SLC45A2 protein is a 530‐amino‐acid polypeptide that contains 12 putative transmembrane domains, and appears to be a transporter that mediates melanin synthesis. Eighteen pathological mutations have been reported so far. In this study, six novel mutations, p.Y49C (c.146A > G), p.G89R (c.265G > A), p.C229Y (c.686G > A), p.T437A (c.1309A > G), p.T440A (c.1318A > G) and p.G473D (c.1418G > A) were found in eight Japanese patients with various clinical phenotypes. The phenotypes of OCA4 were as various as the other types of OCA and probably depended on the mutation sites in the SLC45A2 gene.     

SLC45A2 mutation frequency in Oculocutaneous Albinism Italian patients doesn't differ from other European studies

Background

Oculocutaneous Albinism (OCA) is a heterogeneous group of inherited diseases involving hair, skin and eyes. To date, six forms are recognized on the effects of different melanogenesis genes.OCA4 is caused by mutations in SLC45A2 showing a heterogeneous phenotype ranging from white hair, blue irides and nystagmus to brown/black hair, brown irides and no nystagmus. The high clinic variety often leads to misdiagnosis.Our aim is to contribute to OCA4 diagnosis defining SLC45A2 genetic variants in Italian patients with OCA without any TYR, OCA2 and TYRP1 gene defects.

Materials and methods

After the clinical diagnosis of OCA, all patients received genetic counseling and genetic test. Automatic sequencing of TYR, OCA2, and TYRP1 genes was performed on DNA of 117 albino patients. Multiplex Ligation-dependent Probe Amplification (MLPA) was carried out on TYR and OCA2 genes to increase the mutation rate. SLC45A2 gene sequencing was then executed in the patients with a single mutation in one of the TYR, OCA2, TYRP1 genes and in the patients, which resulted negative at the screening of these genes.

Results

SLC45A2 gene analysis was performed in 41 patients and gene alterations were found in 5 patients. Four previously reported SLC45A2 mutations were found: p.G100S, p.W202C, p.A511E and c.986delC, and three novel variants were identified: p.M265L, p.H94D, and c.1156+1G>A. All the alterations have been detected in the group of patients without mutations in the other OCA genes.

Conclusions

Three new variants were identified in OCA4 gene; the analysis allowed the classification of a patient previously misdiagnosed as OA1 because of skin and hair pigmentation presence. The molecular defects in SLC45A2 gene represent the 3.4% in this cohort of Italian patients, similar to other Caucasian populations; our data differ from those previously published by an Italian researcher group, obtained on a smaller cohort of patients.     

Evidence of digenic inheritance in autoinflammation-associated genes

Familial Mediterranean fever (FMF) has traditionally been considered as a monogenic autosomal recessive disorder caused by mutations in the MEFV gene with highest incidence among Mediterranean populations. In a considerable number of patients with typical FMF, only one MEFV mutation was identified and the possibility that more than one autoinflammatory gene may be responsible for their disease was investigated. In the present study, an extensive search for possible mutations in three hereditary recurrent fever (HRF) genes was performed in 128 MEFV heterozygous Greek–Cypriots clinically diagnosed based on their phenotype with FMF-like disease from a previous study. Sequence analysis was performed for MVK, TNFRSF1A and NLRP3 genes which is also known to cause HRFs. In total, three patients were identified with heterozygous mutations and a second mutation in an autoinflammatory gene. Two patients carried a MEFV mutation and a NLRP3 mutation, and an additional third carried a MEFV mutation and a TNFRSF1A mutation. Patient 1 carried MEFV p.[Val726Ala] (NM_000243.2:c.2177T >C) and NLRP3 p.[Val198Met] (NM_001243133.1:c.592G >A) variants and patient 2 carried MEFV p.[Glu148Gln] (NM_000243.2:c.442G >C) variant which is of uncertain significance and NLRP3 p.[Arg176Trp] (NM_001243133.1:c.526C >T). Lastly, patient 3 was identified to carry MEFV p.[Met694Val] (NM_000243.2:c.2080A >G) and TNFRSF1A p.[Arg121Gln] (NM_001065.3:c.362G >A) variants. The results from this study indicate that screening of genes known to cause HRFs in patients already identified with a single MEFV mutation, can reveal quite rare but potentially causative mutational combinations at different loci. Such interaction provide further evidence for possible locus–locus interactions and phenotypes resulting from digenic inheritance.     

Oculocutaneous Albinism Type 3 (OCA3): Analysis of Two Novel Mutations in <Emphasis Type="Italic">TYRP1</Emphasis> Gene in Two Chinese Patients

Oculocutaneous albinism (OCA) is a genetic disease characterized by the reduction or deficiency of melanin in eyes, skin, and hair. OCA exhibits genetic heterogeneity. Presently, there are four types of OCA named as OCA1, OCA2, OCA3, and OCA4. OCA3 is more common in African born blacks but rarely found in other ethnic populations. Our recent genotyping of patients with OCA of Chinese descent has identified two patients who were not OCA1, OCA2, or OCA4. Examination and analysis of the TYRP1 gene identified them to be having OCA3. PCR and DNA sequencing analysis found that the mutant TYPR1 alleles were present in each of the two patients, c.780-791del/c.1067G>A (p.R356Q) and c.625G>TT (p.G209LfsX1)/c.643C>T (p.H215Y). The c.780-791del and c.1067G>A mutations have been already reported. However, the c.625G>TT and c.643C>T mutations have not been previously reported and were found to be maternal and paternal mutations, respectively. Moreover, population screening and bioinformatic analysis were carried out to determine the effects of these two mutations which revealed that both the mutation were pathogenic. Based on the similar mild phenotype of these two patients, we suggest that OCA3 might be prevalent within the Chinese population.     

Two Novel Tyrosinase (TYR) Gene Mutations with Pathogenic Impact on Oculocutaneous Albinism Type 1 (OCA1)

Oculocutaneous albinism (OCA) is a heterogeneous group of autosomal recessive disorders resulting from mutations of the tyrosinase (TYR) gene and presents with either complete or partial absence of pigment in the skin, hair and eyes due to a defect in an enzyme involved in the production of melanin. In this study, mutations in the TYR gene of 30 unrelated Iranian OCA1 patients and 100 healthy individuals were examined using PCR-sequencing. Additionally, in order to predict the possible effects of new mutations on the structure and function of tyrosinase, these mutations were analyzed by SIFT, PolyPhen and I-Mutant 2 software. Here, two new pathogenic p.C89S and p.H180R mutations were detected in two OCA1 patients. Moreover, the R402Q and S192Y variants, which are common non-pathogenic polymorphisms, were detected in 17.5% and 35% of the patients, respectively. The outcome of this study has extended the genotypic spectrum of OCA1 patients, which paves the way for more efficient carrier detection and genetic counseling.     

Association of five SNPs with human hair colour in the Polish population

Twenty-two variants (single nucleotide polymorphisms – SNPs) of the genes involved in hair pigmentation (OCA2, HERC2, MC1R, SLC24A5, SLC45A2, TPCN2, TYR, TYRP1) were genotyped in a group of 186 Polish participants, representing a range of hair colours (45 red, 64 blond, 77 dark). A genotype-phenotype association analysis was performed.Using z-statistics we identified three variants highly associated with different hair colour categories (rs12913832:A>G in HERC2, rs1805007:T>C and rs1805008:C>T in MC1R). Two variants: rs1800401:C>T in OCA2 and rs16891982:C>G in SLC45A2 showed a high probability of a relation with hair colour, although that probability did not exceed the threshold of statistical significance after applying the Bonferroni correction. We created and validated mathematical logistic regression models in order to test the usefulness of the sets of polymorphisms for hair colour prediction in the Polish population. We subjected four models to stratified cross-validation. The first model consisted of three polymorphisms that proved to be important in the associative analysis. The second model included, apart from the mentioned polymorphisms, additionally rs16891982:C>G in SLC45A. The third model included, apart from the variants relevant in the associating analysis, rs1800401:C>T in OCA. The fourth model consisted of the set of polymorphisms from the first model supplemented with rs16891982:C>G in SLC45A and rs1800401:C>T in OCA. The validation of our models has shown that the inclusion of rs16891982:C>G in SLC45A and rs1800401:C>T in OCA increases the prediction of red hair in comparison with the algorithm including only rs12913832:A>G in HERC2, rs1805007:T>C and rs1805008:C>T in MC1R. The model consisting of all the five above-mentioned genetic variants has shown good prediction accuracies, expressed by the area under the curve (AUC) of the receiver operating characteristics: 0.84 for the red-haired, 0.82 for the dark-haired and 0.71 for the blond-haired.A genotype-phenotype association analysis brought results similar to those in other studies and confirmed the role of rs16891982:C>G, rs12913832:A>G, rs1805007:T>C and rs1805008:C>T in hair colour determination in the Polish population. Our study demonstrated for the first time the possibility of a share of the rs1800401:C>T SNP in the OCA2 gene in hair colour determination. Including this single nucleotide polymorphism in the actual hair colour predicting models would improve their predictive accuracy.     

Aberrant splicing in MLH1 and MSH2 due to exonic and intronic variants

Single base substitutions in DNA mismatch repair genes which are predicted to lead either to missense or silent mutations, or to intronic variants outside the highly conserved splicing region are often found in hereditary nonpolyposis colorectal cancer (HNPCC) families. In order to use the variants for predictive testing in persons at risk, their pathogenicity has to be evaluated. There is growing evidence that some substitutions have a detrimental influence on splicing. We examined 19 unclassified variants (UVs) detected in MSH2 or MLH1 genes in patients suspected of HNPCC for expression at RNA level. We demonstrate that 10 of the 19 UVs analyzed affect splicing. For example, the substitution MLH1,c.2103G>C in the last position of exon 18 does not result in a missense mutation as theoretically predicted (p.Gln701His), but leads to a complete loss of exon 18. The substitution MLH1,c.1038G>C (predicted effect p.Gln346His) leads to complete inactivation of the mutant allele by skipping of exons 10 and 11, and by activation of a cryptic intronic splice site. Similarly, the intronic variant MLH1,c.306+2dupT results in loss of exon 3 and a frameshift mutation due to a new splice donor site 5 bp upstream. Furthermore, we confirmed complete exon skipping for the mutations MLH1,c.1731G>A and MLH1,c.677G>A. Partial exon skipping was demonstrated for the mutations MSH2,c.1275A>G, MLH1,c.588+5G>A, MLH1,c.790+4A>G and MLH1,c.1984A>C. In contrast, five missense mutations (MSH2,c.4G>A, MSH2,c.2123T>A, MLH1,c.464T>G, MLH1,c.875T>C and MLH1,c.2210A>T) were found in similar proportions in the mRNA as in the genomic DNA. We conclude that the mRNA examination should precede functional tests at protein level. Databases: HNPCC – OMIM 114500, MSH2 – OMIM: 120435; GenBank: NM_000251.1, MLH1 – OMIM: 120436; GenBank: NM_000249.2, InSiGHT mutation database: , Programs: BDGP: , ESEfinder program:     

Oculocutaneous albinism type 4: six novel mutations in the membrane-associated transporter protein gene and their phenotypes

Oculocutaneous albinism type 4 (OCA4) is an autosomal recessive hypopigmentary disorder caused by mutations in the Membrane-Associated Transporter Protein gene (SLC45A2). The SLC45A2 protein is a 530-amino-acid polypeptide that contains 12 putative transmembrane domains, and appears to be a transporter that mediates melanin synthesis. Eighteen pathological mutations have been reported so far. In this study, six novel mutations, p.Y49C (c.146A > G), p.G89R (c.265G > A), p.C229Y (c.686G > A), p.T437A (c.1309A > G), p.T440A (c.1318A > G) and p.G473D (c.1418G > A) were found in eight Japanese patients with various clinical phenotypes. The phenotypes of OCA4 were as various as the other types of OCA and probably depended on the mutation sites in the SLC45A2 gene.     

Comprehensive analysis of spectral distribution of a large cohort of Chinese patients with non‐syndromic oculocutaneous albinism facilitates genetic diagnosis

Non‐syndromic oculocutaneous albinism (nsOCA) is a group of genetically heterogeneous autosomal recessive disorders with complete lack or decrease pigmentation in skin, hair, and eyes. TYR, OCA2, TYRP1, SLC45A2, SLC24A5, and LRMDA were reported to cause OCA1‐4 and OCA6‐7, respectively. By sequencing all the known nsOCA genes in 114 unrelated Chinese nsOCA patients combined with In silico analyses, splicing assay, and classification of variants according to the standards and guidelines of American College of Medical Genetics and Genomics, we detected seventy‐one different OCA‐causing variants separately in TYR, OCA2, SLC45A2, and SLC24A5, including thirty‐one novel variants (13 in TYR, 11 in OCA2, and 7 in SLC45A2). This study shows that OCA1 is the most common (75/114) and OCA2 ranks the second most common (16/114) in Chinese. 99 patients of our cohort were caused by variants of all the known nsOCA genes. Cutaneous phenotypes of OCA1, OCA2, and OCA4 patients were shown in this study. The second OCA6 case in China was identified here. These data expand the spectrum of OCA variants as well phenotype and facilitate clinical implement of Chinese OCA patients.     

A 4‐bp deletion promoter variant (rs984225803) is associated with mild OCA4 among Japanese patients

Oculocutaneous albinism (OCA) type 4 is one of the most common types of albinism among Japanese population. In some patients who were clinically diagnosed with OCA, we have found a heterozygous pathological mutation in the coding region of SLC45A2, the gene responsible for OCA4, not leading to a DNA‐based diagnosis. In this study, we evaluated pathological variants in the promoter region of SLC45A2 in these patients. The results indicated that the majority of the patients had a 4‐bp deletion in the said region (c.‐492_489delAATG; GenBank accession number: NM_016180 ; rs984225803) in the contralateral allele. These patients displayed a mild phenotype, especially regarding eye manifestations. The results of the luciferase reporter assay and electrophoretic mobility shift assay supported the pathological role of the variant. In addition, four of 220 alleles in Japanese normal control subjects also showed the deletion variant, indicating that this variant could possibly be a skin color‐associated variant.     

Eight novel CARD15 variants detected by DNA sequence analysis of the CARD15 gene in 111 patients with inflammatory bowel disease

We performed a limited DNA sequence analysis of the CARD15 gene in 89 patients with Crohn’s disease (CD), 19 patients with ulcerative colitis (UC), and three patients with indeterminate colitis (IC), who were heterozygous carriers of one of the common CARD15 mutations [c.2104C>T (p.R702W), c.2722G>C (p.G908R), or c.3019_3020insC (p.Leu1007fsX1008)], the c.2462+10A>C variant, or of a new amino acid substitution in the 3′-end of exon 4. CARD15 exons 4, 5, 6, 8, and 11 were amplified by PCR and completely sequenced, thereby theoretically covering 73.9% of the described CARD15 variants and 96.6% of the mutated alleles. Using this approach, eight novel amino acid substitutions [c.1171C>T (p.R391C), c.1387C>G (p.P463A), c.2138G>A (p.R713H), c.2278C>T (p.R760C), c.2368C>T (p.R790W), c.2371C>T (p.R791W), c.2475C>G (p.N825K), and c.2546C>T (p.A849V)] were detected in six CD and two IC patients, and one UC patient. A severe disease phenotype was observed especially in patients who are compound-heterozygous for a common and a novel CARD15 mutation.Schnitzler and Brand contributed equally     

Missense mutations in the BMP15 gene are associated with ovarian failure

Premature ovarian failure (POF) is an unexplained amenorrhoea (>6 months) with raised levels of gonadotropins (FSH>40 U/L) occurring before the age of 40 years. Recent studies have elucidated the role of oocyte derived growth factors (BMP15 and GDF9) in maintenance of folliculogenesis, granulosa cell (GC) proliferation and overall fertility. Our recently published work showed presence of two rare missense variants in the GDF9 gene associated with ovarian failure (Dixit et al. 2005, Menopause 12:749–754). The present case-control study has been structured to establish the role of BMP15 germline status associated with ovarian failure. Sequence analysis of the coding region of the BMP15 gene was carried out in a cohort of women with POF (n=133), primary amenorrhoea (n=60), and secondary amenorrhoea (n=9) compared with control females (n=197). This study revealed a total of 18 germline variants in the coding region of BMP15 gene, including 16 novel variants. These novel variants include one intronic variant, one 3’ flanking variant, one silent variant, and 13 missense variants. Eleven missense variants were present only in cases with complete absence in the control females. The remaining two missense variants viz. c.308A>G (p.Asn103Ser) and c.788_789insTCT (p.Leu263_Arg264insLeu) were present both in the cases and in the controls. The c.788_789insTCT variant was significantly higher in primary amenorrhoea cases than in the controls (Fisher’s exact test, P=0.034). Three frequent variants c.-9C>G, c.308A>G, and c.852C>T were chosen for haplotyping. The haplotype G-G-C was found to be significantly associated with ovarian failure (P=0.0075). In a nutshell, the BMP15 gene is highly associated with etiology of ovarian failure.     

Mutational analysis of TYR gene and its structural consequences in OCA1A

Oculocutaneous albinism type 1A (OCA1A) is the most severe form of albinism characterized by a complete lack of melanin production throughout life and is caused by mutations in the TYR gene. TYR gene codes tyrosinase protein to its relation with melanin formation by knowing the function of these SNPs. Based on the computational approaches, we have analyzed the genetic variations that could change the functional behaviour by altering the structural arrangement in TYR protein which is responsible for OCA1A. Consequences of mutation on TYR structure were observed by analyzing the flexibility behaviour of native and mutant tyrosinase protein. Mutations T373K, N371Y, M370T and P313R were suggested as high deleterious effect on TYR protein and it is responsible for OCA1A which were also endorsed with previous in vivo experimental studies. Based on the quantitative assessment and flexibility analysis of OCA1A variants, T373K showed the most deleterious effect. Our analysis determines that certain mutations can affect the dynamic properties of protein and can lead to disease conditions. This study provides a significant insight into the underlying molecular mechanism involved in albinism associated with OCA1A.     

Genetic analysis of <Emphasis Type="Italic">COL11A2</Emphasis> in Korean patients with autosomal dominant non-syndromic hearing loss

The collagen type XI alpha 2 gene (COL11A2) is associated with autosomal dominant non-syndromic hearing loss (ADNSHL), and all mutations of this gene in ADNSHL are missense mutations. To evaluate its potential as a major causative gene of ADNSHL in the Korean population, we performed genetic analysis of COL11A2 in 75 unrelated Korean patients with ADNSHL. Consequently, 5 non-synonymous variants, 7 synonymous variants, and 6 intronic variants were identified in COL11A2. Among them, a novel variant, p.G829R (c.2485G>C) was found in a patient as a heterozygote. However, pedigree analysis showed this variation was not co-segregated with hearing loss. Previously reported variants p.G230W (c.688G>T) and p.P1422L (c.4265C>T) were discovered in Korean patients. However, these variants were also detected in normal individuals. These results suggest that COL11A2 is not a major causative gene of ADNSHL in the Korean population.     

ARSB gene variants causing Mucopolysaccharidosis VI in Miniature Pinscher and Miniature Schnauzer dogs

Mucopolysaccharidosis (MPS) VI is a lysosomal storage disease caused by a deficiency of N-acetylgalactosamine-4-sulfatase, also called arylsulfatase B (ARSB, EC 3.1.6.12). Dogs with MPS VI show progressive predominantly oculoskeletal signs homologous to those in human and feline patients. We report herein two pathogenic ARSB gene variants in Miniature Pinscher and Miniature Schnauzer dogs with MPS VI and a genotyping survey in these breeds. All exons and adjacent regions of the ARSB gene were sequenced from three affected Miniature Pinschers and three affected Miniature Schnauzers. Allelic discrimination assays were used for genotyping. A missense variant (NM_001048133.1:c.910G>A) was found in exon 5 of MPS VI-affected Miniature Pinschers that is predicted to result in a deleterious amino acid substitution of a highly conserved glycine to arginine (NP_001041598.1:p.Gly304Arg). In MPS VI-affected Miniature Schnauzers, a 56 bp deletion (NM_001048133.1:c.-24_32del) was found at the junction of exon 1 and its upstream region, predicting no enzyme synthesis. All clinically affected Miniature Pinschers and Miniature Schnauzers were homozygous for the respective variants, and screened healthy dogs in each breed were either heterozygous or homozygous for the wt allele. Whereas the Miniature Pinscher variant seemed to occur commonly (0.133 allele frequency), the Miniature Schnauzer variant was presumed to be rare. In conclusion, two breed-specific pathogenic ARSB gene variants were identified in Miniature Pinscher and Miniature Schnauzer dogs with MPS VI, allowing for genotyping and informed breeding to prevent the production of affected offspring.