# MITF p.Arg217Thr Variant Identified in a Han Chinese Family with Tietz/Waardenburg Syndrome

Authors: Rong Yu; Lv Liu; Ya-Li Li; Liang-Liang Fan
Journal: BioMed Research International
 (2021)

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## Abstract

Waardenburg syndrome (WS) is a group of rare genetic disorders characterized by hearing loss, changes in coloring of hair, skin, and eyes, and alterations in the shape of the face. Tietz syndrome is another rare disorder which presented similar phenotypes to WS. Patients with Tietz/Waardenburg syndrome often present with pale blue eyes, albino skin, and distinctive hair coloring, such as a patch of white hair or hair that prematurely turns gray. At present, more than six candidate genes are responsible for four types of Waardenburg syndrome and Tietz syndrome. This study is aimed at identifying the pathogenic gene variants in a three-generation Han Chinese family with hearing loss, blue-gray iris, albino skin, and white hair. In order to discover the molecular genetic lesion underlying the disease phenotype, whole exome sequencing in the proband, with Tietz/Waardenburg syndrome phenotypes, of a Han Chinese family from HeBei, China, was conducted. A novel heterozygous c.650G>C/p.Arg217Thr variant inmelanocyte inducing transcription factor (MITF) was identified. Sanger sequencing further validated that this mutation existed in three affected individuals and absent in healthy family members. Bioinformatics analysis predicted that this mutation was deleterious. Our study further identified the genetic lesion of the family. Simultaneously, our study may also contribute to genetic counseling, embryonic screening of in vitro fertilized embryos, and prenatal genetic diagnosis of patients with Tietz/Waardenburg syndrome, especially for the proband, unmarried and unpregnant women, to reduce familial transmission in this Han Chinese family.


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## Body

## 1. Introduction

Waardenburg syndrome (WS) represents several rare genetic disorders that cause hearing loss, changes in coloring of hair, skin, and eyes, and alterations in the shape of the face [1, 2]. The typical inherited pattern of WS is autosomal dominant trait with genetic heterogeneity [3, 4]. It is estimated that the prevalence of WS is approximately 1/42,000 globally, and in persons with deaf-mutism, the syndrome is observed from 0.9% to 2.8% [5]. Patients with WS show the pale blue eyes or different colored eyes, as well as distinctive hair coloring, such as a patch of white hair or hair that prematurely turns gray [1–4]. Tietz syndrome is another rare disorder which presented similar phenotypes to WS [6], such as congenital hearing loss, albino skin, and blue iris.The previous studies demonstrated that the melanocytes, one type of pigment-producing cells, participated in the formation and development of Tietz/Waardenburg syndrome [7, 8]. Melanocytes produce a pigment called melanin, which contributes to skin, hair, and eye color and plays a crucial role in the normal function of the inner ear [4, 9]. Variants in at least six genes including endothelin 3 (EDN3), endothelin receptor type B (EDNRB), melanocyte inducing transcription factor (MITF), paired box 3 (PAX3), snail family transcriptional repressor 2 (SNAI2), and SRY-box transcription factor 10 (SOX10) may disrupt the normal development of melanocytes, resulting in abnormal pigmentation of the skin, hair, and eyes and hearing function [7, 10]. In addition, recently, some studies also indicated that nontruncating mutation of MITF basic domain is associated with Tietz syndrome [6].In this context, a heterozygous mutation (NT_022495: c.650G>C/p.Arg217Thr) ofMITF was identified via employing whole exome sequencing and Sanger sequencing in a Han Chinese family with hearing loss, blue-gray iris, albino skin, and white hair. It may be the genetic etiology for this family and have critical implications for genetic monitoring.


## 2. Materials and Methods

### 2.1. Pedigrees and Participators

A 16-person, three-generation Han Chinese pedigree was recruited at HeBei General Hospital, Shijiazhuang, China (Figure1(a)). Clinical data and peripheral blood samples were obtained from 15 members, including three affected (II-1, II-9, and III-1) and 12 unaffected members. Simultaneously, 200 unrelated local healthy people were also enrolled to serve as normal controls. All the subjects have provided written informed consent, and the research project was approved by the ethics committee of HeBei General Hospital.Figure 1
The clinical data of the family with Tietz/Waardenburg syndrome. (a) Pedigree figure. Squares, male family members; circles, female members; arrow, proband. The (b) white hair, (c) blue-gray iris, and (d) albino skin of the proband. (e) The audiometry showed the proband suffered from sensorineural hearing loss. ASSR: audio steady-state response; PTA: pure-tone audiometry.
(a)(b)(c)(d)(e)


### 2.2. Whole Exome Sequencing

Genomic DNA was prepared from peripheral blood of the patients and all other participants using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) as we have described [11]. The proband was selected to perform whole exome sequencing (WES). Exome capture and high-throughput sequencing were performed in Novogene Bioinformatics Institute (Beijing, China). One microgram of qualified genomic DNA from the proband was captured with the Agilent’s SureSelect Human All Exon kit V5 (Agilent Technologies, Inc., Santa Clara, USA) and sequenced by Illumina Hiseq 4000 (Illumina Inc., San Diego, USA). Briefly, genomic DNA was randomly sheared by Covaris S220 sonicator (Covaris, Inc., Woburn, USA). Then, the fragments of DNAs were subjected to three enzymatic steps: end repair, A-tailing, and adapter ligation. The adapter-ligated DNA fragments were amplified with Herculase II Fusion DNA Polymerase (Agilent). Finally, the precapture libraries containing exome sequences were captured using SureSelect capture library kit (Agilent). After DNA quality assessment, the captured DNA library underwent high-throughput sequencing on Illumina Hiseq 4000 platform. Downstream processing was performed using the Genome Analysis Toolkit (GATK), Varscan2, and Picard, and variant calls were made with the GATK HaplotypeCaller. Variant annotation was based on Ensembl release 82, and filtering was performed with ANNOVAR Documentation.Nonsynonymous SNPs or frameshift-causing INDELs with an alternative allelefrequency>0.005 in the NHLBI Exome Sequencing Project Exome Variant Server (ESP6500), dbSNP138 (https://www.ncbi.nlm.nih.gov/projects/SNP/index.html), the 1000 Genomes project (https://www.1000genomes.org/), the ExAC database (http://exac.broadinstitute.org), or in-house exome databases of Novogene (2500 exomes) were excluded prior to analysis. Then, the filtered SNVs and INDELs, predicted by HapMap Genome Browser (https://hapmap.ncbi.nlm.nih.gov/), SIFT (http://sift.jcvi.org/), and MutationTaster (http://www.mutationtaster.org/) to be nondeleterious, were excluded. In addition, we paid close attention to mutations in albino skin-related genes (Table S1).


### 2.3. Variant Validation and Cosegregation Analysis

Variant validation and cosegregation analysis were performed on each member by Sanger sequencing with the following primers ofMITF (NT_022495, NM_000248, and NP_000239) and designed by Primer3: 5-′TTCCGTTGTCATGACCTGGA-3′ and 5-′AACACGCGATTGTACTCACG-3′. The candidate variant was also examined in 200 healthy adults of both sexes and different ages, who were enrolled by ourselves and to be used as an internal control for genetic variants potentially specific for the Han Chinese [12].


## 2.1. Pedigrees and Participators

A 16-person, three-generation Han Chinese pedigree was recruited at HeBei General Hospital, Shijiazhuang, China (Figure1(a)). Clinical data and peripheral blood samples were obtained from 15 members, including three affected (II-1, II-9, and III-1) and 12 unaffected members. Simultaneously, 200 unrelated local healthy people were also enrolled to serve as normal controls. All the subjects have provided written informed consent, and the research project was approved by the ethics committee of HeBei General Hospital.Figure 1
The clinical data of the family with Tietz/Waardenburg syndrome. (a) Pedigree figure. Squares, male family members; circles, female members; arrow, proband. The (b) white hair, (c) blue-gray iris, and (d) albino skin of the proband. (e) The audiometry showed the proband suffered from sensorineural hearing loss. ASSR: audio steady-state response; PTA: pure-tone audiometry.
(a)(b)(c)(d)(e)


## 2.2. Whole Exome Sequencing

Genomic DNA was prepared from peripheral blood of the patients and all other participants using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) as we have described [11]. The proband was selected to perform whole exome sequencing (WES). Exome capture and high-throughput sequencing were performed in Novogene Bioinformatics Institute (Beijing, China). One microgram of qualified genomic DNA from the proband was captured with the Agilent’s SureSelect Human All Exon kit V5 (Agilent Technologies, Inc., Santa Clara, USA) and sequenced by Illumina Hiseq 4000 (Illumina Inc., San Diego, USA). Briefly, genomic DNA was randomly sheared by Covaris S220 sonicator (Covaris, Inc., Woburn, USA). Then, the fragments of DNAs were subjected to three enzymatic steps: end repair, A-tailing, and adapter ligation. The adapter-ligated DNA fragments were amplified with Herculase II Fusion DNA Polymerase (Agilent). Finally, the precapture libraries containing exome sequences were captured using SureSelect capture library kit (Agilent). After DNA quality assessment, the captured DNA library underwent high-throughput sequencing on Illumina Hiseq 4000 platform. Downstream processing was performed using the Genome Analysis Toolkit (GATK), Varscan2, and Picard, and variant calls were made with the GATK HaplotypeCaller. Variant annotation was based on Ensembl release 82, and filtering was performed with ANNOVAR Documentation.Nonsynonymous SNPs or frameshift-causing INDELs with an alternative allelefrequency>0.005 in the NHLBI Exome Sequencing Project Exome Variant Server (ESP6500), dbSNP138 (https://www.ncbi.nlm.nih.gov/projects/SNP/index.html), the 1000 Genomes project (https://www.1000genomes.org/), the ExAC database (http://exac.broadinstitute.org), or in-house exome databases of Novogene (2500 exomes) were excluded prior to analysis. Then, the filtered SNVs and INDELs, predicted by HapMap Genome Browser (https://hapmap.ncbi.nlm.nih.gov/), SIFT (http://sift.jcvi.org/), and MutationTaster (http://www.mutationtaster.org/) to be nondeleterious, were excluded. In addition, we paid close attention to mutations in albino skin-related genes (Table S1).


## 2.3. Variant Validation and Cosegregation Analysis

Variant validation and cosegregation analysis were performed on each member by Sanger sequencing with the following primers ofMITF (NT_022495, NM_000248, and NP_000239) and designed by Primer3: 5-′TTCCGTTGTCATGACCTGGA-3′ and 5-′AACACGCGATTGTACTCACG-3′. The candidate variant was also examined in 200 healthy adults of both sexes and different ages, who were enrolled by ourselves and to be used as an internal control for genetic variants potentially specific for the Han Chinese [12].


## 3. Results

### 3.1. Pedigree and Clinical Characteristics

The proband (III-1), a 27-year-old woman, presented with white hair (Figure1(b)), blue-gray iris (Figure 1(c)), albino skin (Figure 1(d)), hearing loss (Figure 1(e)), and flecking over the face and hand. Medical history survey revealed that the proband presented with white hair at six years of age, and two years later, the face and extremity showed prominent flecking. At ten, the proband presented with albino skin. According to the family member’s memories, the proband was diagnosed as having congenital hearing loss at the age of three. The examination of external auditory canal and eardrum was normal, and the results of pure-tone audiometry (PTA) and audio steady-state response (ASSR) of the proband are in agreement, indicating severe-to-profound hearing loss (Figure 1(e)). Physical examination showed blue-gray iris with normal vision (left 1.0 and right 1.2), inner canthal diameter of 3.4 cm, interpupillary distance of 6.5 cm, outer canthal diameter of 9.0 cm, and W index of 1.77 (normal rage: <1.95). Meanwhile, the proband also showed tears after light stimulation. Family history investigation indicated that the proband’s father (II-1) also presented with white hair, blue-gray iris, albino skin, and hearing loss, but one of the proband’s uncles (II-9) showed gray hair, one eye with blue iris and the other eye with brown iris (or heterochromia), and unilateral hearing loss (right side). In addition, according to the description of the proband, her grandfather (I-1) presented white hair as well. All the clinical symptoms of the affected family members are summarized in Table 1.Table 1
Clinical features of the family with Tietz/Waardenburg syndrome.
FeaturesIII-1II-1II-9I-2Age274938DiedGenderFMMMSkinAlbino skinAlbino skinUnknownUnknownHairWhite hairWhite hairGray hairWhite hairEyeBlue-gray irisBlue-gray irisOne eye with blue iris and one eye with brown irisUnknownEarHearing lossHearing lossRight side hearing lossUnknownF: female; M: male.


### 3.2. Genetic Analysis

The mean coverage of the target regions obtained for the proband was 99.8%, with average sequencing depth of 89.47×. In total, 10,271 SNPs and 15,147 INDELS were identified. Via abovementioned filtering method, a heterozygous c.650G>C/p.Arg217Thr variant inMITF was identified. No other potential pathogenic mutations for hearing loss and/or albinism skin were found. The mutation was validated by Sanger sequencing and was also detected in another two affected family members (II-1 and II-9) (Figure 2(a)). In addition, the variant c.650G>C/p.Arg217Thr was absent in other healthy family individuals (Figure 2(a)) and 200 unrelated Han Chinese healthy controls and other public databases, such as Exome Aggregation Consortium database (ExAC) and Genome Aggregation Database (gnomAD). Bioinformatics programs predicated that this novel (c.650G>C/p.Arg217Thr) mutation was disease causing and located in an evolutionarily conserved site of MITF protein (Figure 2(b)). According to ACMG guideline [13], this novel mutation belongs to likely pathogenic criteria (PM2+PM5+PP1+PP3).Figure 2
The genetic analysis of the family with Tietz/Waardenburg syndrome. (a) Sequencing results of theMITF mutation. Sequence chromatogram indicates a G to C transition of nucleotide 650. (b) Alignment of multiple MITF protein sequences across species. The Arg217 affected amino acid locates in the highly conserved amino acid region in different mammals (from Ensembl). Red word shows the Arg217 site.
(a)(b)


## 3.1. Pedigree and Clinical Characteristics

The proband (III-1), a 27-year-old woman, presented with white hair (Figure1(b)), blue-gray iris (Figure 1(c)), albino skin (Figure 1(d)), hearing loss (Figure 1(e)), and flecking over the face and hand. Medical history survey revealed that the proband presented with white hair at six years of age, and two years later, the face and extremity showed prominent flecking. At ten, the proband presented with albino skin. According to the family member’s memories, the proband was diagnosed as having congenital hearing loss at the age of three. The examination of external auditory canal and eardrum was normal, and the results of pure-tone audiometry (PTA) and audio steady-state response (ASSR) of the proband are in agreement, indicating severe-to-profound hearing loss (Figure 1(e)). Physical examination showed blue-gray iris with normal vision (left 1.0 and right 1.2), inner canthal diameter of 3.4 cm, interpupillary distance of 6.5 cm, outer canthal diameter of 9.0 cm, and W index of 1.77 (normal rage: <1.95). Meanwhile, the proband also showed tears after light stimulation. Family history investigation indicated that the proband’s father (II-1) also presented with white hair, blue-gray iris, albino skin, and hearing loss, but one of the proband’s uncles (II-9) showed gray hair, one eye with blue iris and the other eye with brown iris (or heterochromia), and unilateral hearing loss (right side). In addition, according to the description of the proband, her grandfather (I-1) presented white hair as well. All the clinical symptoms of the affected family members are summarized in Table 1.Table 1
Clinical features of the family with Tietz/Waardenburg syndrome.
FeaturesIII-1II-1II-9I-2Age274938DiedGenderFMMMSkinAlbino skinAlbino skinUnknownUnknownHairWhite hairWhite hairGray hairWhite hairEyeBlue-gray irisBlue-gray irisOne eye with blue iris and one eye with brown irisUnknownEarHearing lossHearing lossRight side hearing lossUnknownF: female; M: male.


## 3.2. Genetic Analysis

The mean coverage of the target regions obtained for the proband was 99.8%, with average sequencing depth of 89.47×. In total, 10,271 SNPs and 15,147 INDELS were identified. Via abovementioned filtering method, a heterozygous c.650G>C/p.Arg217Thr variant inMITF was identified. No other potential pathogenic mutations for hearing loss and/or albinism skin were found. The mutation was validated by Sanger sequencing and was also detected in another two affected family members (II-1 and II-9) (Figure 2(a)). In addition, the variant c.650G>C/p.Arg217Thr was absent in other healthy family individuals (Figure 2(a)) and 200 unrelated Han Chinese healthy controls and other public databases, such as Exome Aggregation Consortium database (ExAC) and Genome Aggregation Database (gnomAD). Bioinformatics programs predicated that this novel (c.650G>C/p.Arg217Thr) mutation was disease causing and located in an evolutionarily conserved site of MITF protein (Figure 2(b)). According to ACMG guideline [13], this novel mutation belongs to likely pathogenic criteria (PM2+PM5+PP1+PP3).Figure 2
The genetic analysis of the family with Tietz/Waardenburg syndrome. (a) Sequencing results of theMITF mutation. Sequence chromatogram indicates a G to C transition of nucleotide 650. (b) Alignment of multiple MITF protein sequences across species. The Arg217 affected amino acid locates in the highly conserved amino acid region in different mammals (from Ensembl). Red word shows the Arg217 site.
(a)(b)


## 4. Discussion

At present, there are four recognized types of WS, which can be distinguished by different clinical features. All four types of WS are present with hearing loss and changes in pigmentation of the hair, skin, and eyes [1, 2]. The clinical features are similar between type 1 and type 2, but the deafness occurs more often in patients with type 2, and people with type 1 almost always have widely spaced eyes [14, 15]. Tietz syndrome also presented similar phenotypes to WS type 2 [6]. Type 3 shows abnormalities of arms and hands in addition to typical features of WS [7]. Type 4 presents symptoms of both WS and Hirschsprung disease, an intestinal disorder with severe constipation or blockage of the intestine [16]. In this study, the proband showed WS features with 1.75 W index which was less than 1.95 (The most significant difference between WS type 1 and type 2 is dystopia canthorum. The W index less than 1.95 was supposed to WS type 2), but the proband also presented with generalized hypopigmentation of skin and eye, which were the features of Tietz syndrome [6]. Hence, the patient may be diagnosed as overlapping Tietz/Waardenburg syndrome. Whole exome sequencing and Sanger sequencing identified a novel mutation (c.650G>C/p.Arg217Thr) of MITF in the proband and other affected members, which further confirmed the clinical diagnosis, because MITF was the pathogenic gene of Tietz/Waardenburg syndrome [10, 17].The humanMITF gene encoding melanocyte inducing transcription factor is located on chromosome 3p13, and it consists of 10 exons, spanning approximately 22.8 kilobases (kb). Previous studies found that MITF, containing both basic helix-loop-helix and leucine zipper structural features, is vital for the development and survival of melanocytes, osteoclasts, and mast cells [17]. Melanocyte development is responsible for pigment cell-specific transcription of the melanogenesis enzyme genes, as well as serves as an amplified oncogene in melanoma [18–20]. Mutations of MITF may affect the survival and differentiation of melanocytes, which may affect the production and distribution of melanin [21] and finally lead to the flecking, generalized hypopigmentation of hair and skin [6]. The leucine zipper structural is responsible for binding identical DNA sequences. In this study, the novel mutation (c.650G>C/p.Arg217Thr) is located in the leucine zipper structural, which may disrupt the stability between MITF and identical DNA sequences and affect the synthesis of enzymes that are essential for melanin production in differentiated melanocytes. Finally, the mutation may disturb the survival and differentiation of melanocytes, which producing melanin to adjust hair, skin, and eye color and the normal function of the inner ear [22, 23].In mice, mutantMITF can lead to deafness, bone hyperdensity, small eyes, and absence of pigment in eyes and skin [24]. Furthermore, MITF mutations, affecting the development of neural crest-derived pigment cells, have been discovered across many species like rat, hamster, and quail [25]. These mutations also affect the development of eyes, whereas only the rat and quail mutations affect osteoclasts. Variants in nacre, a homologous gene of MITF in zebrafish, only affect neural crest melanocytes [26]. In addition, studies of Drosophila showed that Dmel, a homologous gene of MITF, was expressed during embryogenesis and in the eye imaginal disk during development [27]. Studies of these different species demonstrate that MITF is an evolutionarily conserved protein, which is functionally essential for normal melanocytic development.In addition, some studies found that WS type 2 in conjunction with ocular albinism (OA) may result from a digenic mutation mechanism including both aMITF mutation and the TYR(R402Q) hypomorphic allele or TYRP mutation [28, 29]. In our study, the proband presented with Tietz/Waardenburg syndrome phenotypes. However, we checked the sequencing data and did not detect the TYR and TYPR gene mutation. Hence, we believed that the case in our study was only caused by the novel mutation (c.650G>C/p.Arg217Thr) of MITF. Simultaneously, according to the HGMD database, about 70 mutations of MITF have been reported in patients with Tietz/Waardenburg type 2 syndrome, especially the mutation p.Arg217Ile and p.Arg217Gly, which is fairly similar to our mutation, which indicated that the site of p.Arg217 may play a crucial role in the MITF function [17, 30].


## 5. Conclusion

In conclusion, a novel (c.650G>C/p.Arg217Thr) variant ofMITF was identified in a Han Chinese family with Tietz/Waardenburg syndrome. The identification of this MITF c.650G>C mutation may contribute to genetic counseling, embryonic screening of in vitro fertilized embryos, and prenatal genetic diagnosis of patients with Tietz/Waardenburg syndrome, especially for the proband, unmarried and unpregnant women, to reduce familial transmission in this Han Chinese family.



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*Source: 4381272-2021-01-12.xml*