Congenital Sensorineural Hearing Loss and Inborn Pigmentary Disorders: First Report of Multilocus Syndrome in Piebaldism

Congenital sensorineural hearing loss may occur in association with inborn pigmentary defects of the iris, hair, and skin. These conditions, named auditory-pigmentary disorders (APDs), represent extremely heterogeneous hereditary diseases, including Waardenburg syndromes, oculocutaneous albinism, Tietz syndrome, and piebaldism. APDs are part of the neurocristopathies, a group of congenital multisystem disorders caused by an altered development of the neural crest cells, multipotent progenitors of a wide variety of different lineages, including those differentiating into peripheral nervous system glial cells and melanocytes. We report on clinical and genetic findings of two monozygotic twins from a large Albanian family who showed a complex phenotype featured by sensorineural congenital deafness, severe neuropsychiatric impairment, and inborn pigmentary defects of hair and skin. The genetic analyzes identified, in both probands, an unreported co-occurrence of a new heterozygous germline pathogenic variant (c.2484 + 5G > T splicing mutation) in the KIT gene, consistent with the diagnosis of piebaldism, and a heterozygous deletion at chromosome 15q13.3, responsible for the neuropsychiatric impairment. This case represents the first worldwide report of dual locus inherited syndrome in piebald patients affected by a complex auditory-pigmentary multisystem phenotype. Here we also synthesize the clinical and genetic findings of all known neurocristopathies characterized by a hypopigmentary congenital disorder.

Here, we describe clinical features and genetic data of two Caucasian monozygotic twins affected by an unreported neuro-cutaneous phenotype characterized by inborn SNHL, a congenital disorder of pigmentation and severe neuropsychiatric impairment.
The most common OCA type in Africa accounting for 30% of cases worldwide Incidence 1:20,000/40.000 births Generalized congenital hypopigmentation of the skin (never white but range from very fair to near normal). The skin color may darken over time and sun exposure Lightly pigmented hair, eyebrows and eyelashes (never white but range from light yellow to blonde to brown), the hair color may darken with age Extremely rare Generalized congenital hypopigmentation of the skin (never white, range from creamy white to near normal) Lightly pigmented hair, eyebrows and eyelashes (never white but range from silvery to golden or near normal) The hair color may darken with age.
Ocular signs: like OCA1 with visual acuity usually better than OCA1. OCA6 and 7 patients do not present an obvious change in the pigmentation patterns. Iris color: it ranges from blue to brown Note 1 : AD autosomal dominant, AR autosomal recessive. Note 2 : the average penetrance of clinical signs is specified in brackets.

Clinical Findings
The probands, two 21 year old male monozygotic twins (IV-3 and IV-4, Figure 1), were referred to our department for dermatologic evaluation. Clinical examination revealed numerous regularly shaped hypopigmented patches on the face, trunk, and upper and lower extremities, mainly distributed on the frontal side of the body (Figure 2). Several hyperpigmented dots and macules were present within and along the margins of the leukoderma areas. Both probands had blond hair with tufts of leukotrichia on the scalp, eyelids, and eyebrows. Both had blue eyes, but only IV-4 showed heterochromia at the right iris. They did not have dystopia canthorum, nor were they dysmorphic.

Clinical Findings
The probands, two 21 year old male monozygotic twins (IV-3 and IV-4, Figure 1), were referred to our department for dermatologic evaluation. Clinical examination revealed numerous regularly shaped hypopigmented patches on the face, trunk, and upper and lower extremities, mainly distributed on the frontal side of the body (Figure 2). Several hyperpigmented dots and macules were present within and along the margins of the leukoderma areas. Both probands had blond hair with tufts of leukotrichia on the scalp, eyelids, and eyebrows. Both had blue eyes, but only IV-4 showed heterochromia at the right iris. They did not have dystopia canthorum, nor were they dysmorphic.  Patients suffered from profound bilateral congenital SNHL, neurodevelopmental delay, severe intellectual disability, and childhood onset of grafted psychosis. They displayed speech disorders and social isolation.
Probands were the first children of an unrelated couple of Albanian parents, coming from a small community of central Albania. The mother (Figure 1, III-6), a 50 year old woman, reported that her sons' depigmented lesions were congenital and did not evolve. Her dermatological examination also showed inborn hypopigmented skin and hair lesions, similar but less extended than those of her children (Figure 2). None of her family members presented any neurological sign.
We could not perform a dermatological examination on the father, (Figure 1, III-5), a 55 year old man affected by a severe form of adult-onset schizophrenia. His wife reported he did not present any skin or hair anomalies. On the other hand, several males and female relatives of III-6 ( Figure 1) presented similar skin and hair congenital lesions shown in the probands. A dermatological exam of the probands' maternal uncle (III-9) and his 6 month old daughter (IV-6), showed diffuse areas of leukoderma and leukotrichia in the absence of neurological symptoms.
Clinical skin and hair features of the probands suggested a diagnosis of PBT, generally associated with mutations in the KIT gene (MIM *164920, NM_000222). Patients suffered from profound bilateral congenital SNHL, neurodevelopmental delay, severe intellectual disability, and childhood onset of grafted psychosis. They displayed speech disorders and social isolation.
Probands were the first children of an unrelated couple of Albanian parents, coming from a small community of central Albania. The mother (Figure 1, III-6), a 50 year old woman, reported that her sons' depigmented lesions were congenital and did not evolve. Her dermatological examination also showed inborn hypopigmented skin and hair lesions, similar but less extended than those of her children (Figure 2). None of her family members presented any neurological sign.
We could not perform a dermatological examination on the father, (Figure 1, III-5), a 55 year old man affected by a severe form of adult-onset schizophrenia. His wife reported he did not present any skin or hair anomalies. On the other hand, several males and female relatives of III-6 ( Figure 1) presented similar skin and hair congenital lesions shown in the probands. A dermatological exam of the probands' maternal uncle (III-9) and his 6 month old daughter (IV-6), showed diffuse areas of leukoderma and leukotrichia in the absence of neurological symptoms.
Clinical skin and hair features of the probands suggested a diagnosis of PBT, generally associated with mutations in the KIT gene (MIM *164920, NM_000222).

Materials and Methods
Genomic DNA was isolated from peripheral blood using a standard procedure (Qiagen, Hilden, Germany) and quantified by Nanodrop spectrophotometer (Thermo Scientific Waltham, Massachusetts, USA). The KIT gene (NM_000222.2) coding exons were amplified and sequenced using Sanger sequencing.
We performed array-CGH with a 60K whole-genome oligonucleotide microarray following the manufacturer's protocol (Agilent Technologies, Santa Clara, California, USA). Slides were scanned using a G2565BA scanner and analyzed using Agilent CGH Analytics software ver. 4.0.81 (Agilent Technologies) with the statistical algorithm ADM-2 and a sensitivity threshold of 6.0. At least three

Materials and Methods
Genomic DNA was isolated from peripheral blood using a standard procedure (Qiagen, Hilden, Germany) and quantified by Nanodrop spectrophotometer (Thermo Scientific Waltham, Massachusetts, USA). The KIT gene (NM_000222.2) coding exons were amplified and sequenced using Sanger sequencing.
We performed array-CGH with a 60K whole-genome oligonucleotide microarray following the manufacturer's protocol (Agilent Technologies, Santa Clara, California, USA). Slides were scanned using a G2565BA scanner and analyzed using Agilent CGH Analytics software ver. 4.0.81 (Agilent Technologies) with the statistical algorithm ADM-2 and a sensitivity threshold of 6.0. At least three consecutive aberrant probes identified significant copy-number changes. We compared our findings to known CNVs listed in the Database of Genomic Variants (DGV, http://projects.tcag.ca/variation) and in the DECIPHER database (https://decipher.sanger.ac.uk/). TaqMan real-time quantitative PCR (qPCR) analysis was used to measure copy number variants at 3q23 and 15q13.2q13.3 in genomic DNA as follows: (a) 3q23 duplication, GK5 (NM_001039547.2) exon 16, primers 5 -agactggaagctccctgaaa; 5 -tcccacatacatgaaagcaca; #38 UPL probe (Roche Diagnostics); (b) 15q13.2q13.3 deletion, CHRNA7 (NM_000746) exon 2, primers 5 -caatgactcgcaaccactca; 5 -atccacgtccatgatctgc; #7 UPL probe (Roche Diagnostics); and (c) RNaseP reference gene, VIC-labeled pre-designed TaqMan gene expression assays (P/N 4316844, Applied Biosystems). We carried out the reaction with an ABI 7500 Fast real-time PCR machine using the ABI TaqMan Universal PCR master mix according to the manufacturer's instructions (Applied Biosystems, Foster City, USA). Efficiencies of the assays were similar and in a range of 90% to 110%. Samples from affected individuals and unrelated healthy controls were run in triplicate. The mean Ct value was used for calculations using the ∆∆Ct method [16].

Genetic Findings
The sequence of the coding exons and flanking intron sequences of the KIT gene allowed the identification of a heterozygous c.2484+5G>T change in intron 17. The variant was not reported in the Genome Aggregation Database (GnomAD; http://gnomad.broadinstitute.org/), and it was predicted to reduce the score of the donor splice site of exon 17 (MutationTaster, http://www.mutationtaster. org/: pathogenic; Splice Site Prediction by Neural Network, http://www.fruitfly.org/seq_tools/splice.

Discussion
PBT is a rare autosomal dominant genodermatosis caused by mutations in the c-kit proto-oncogene, which encodes the transmembrane receptor tyrosine kinase for mast cell growth factor (MGF, also known as stem cell factor) [9][10][11][12][13][14]. The KIT receptor and its ligand (KITLG) act as crucial factors in the control of physiological and pathological skin pigmentation through the Ras ⁄ mitogen-activated protein kinase (MAPK) signaling pathway. Accordingly, loss-of-function KIT mutations determine defects in the survival, proliferation, differentiation, and migration of melanoblasts from the NC to the skin during early embryonic development. Consequently, patients have a significant or complete loss of melanocytes in the affected areas of the hair and skin [9,10]. PBT is clinically characterized by congenital leukoderma, leukotrichia of hair, eyebrows and eyelashes, and, rarely, heterochromia of irides, with a great variation in the degree and pattern of presentation, even within affected families. Specifically, piebald patients are featured by congenital, well-demarked, symmetrical, non-pigmented white patches involving the skin of the face, trunk, arms, and legs [6,9,10]. They also frequently show poliosis, traditionally known as "white forelock", a localized patch of white hair in a group of hair follicle; it is often triangular in shape and may be the only manifestation of PBT in 80% to 90% of c-Kit carriers. In some cases, both the hair and the underlying forehead may be affected [6,9]. The skin lesions, histologically characterized by the congenital absence of melanocytes, are usually stable during life, although hyperpigmented dots or macules may appear within or at their margins. Sometimes, café-au-lait macules can be present in piebald patients who concomitantly may also be affected by neurofibromatosis type 1 (NF1, MIM *613113) [9,11]. Very few piebald subjects harbor a heterozygous change in the SNAI2 gene. It is located on chromosome 8 at position 11.21 and encodes for SNAIL2, a protein that belongs to the Snail family of zinc finger transcription factors. SNAIL2 is involved in the regulation of differentiation and migration of NC cells during embryonic development [4][5][6]. It is interesting to note that SNAI2 germline mutations in a homozygous state were reported in a few human cases affected by a Waardenburg syndrome type 2 [7].
We describe a large family of Albanian descent harboring a germline pathogenic change in the KIT gene (Figure 1). Three subjects carrying the c.2484 + 5G > T splicing mutation presented skin and hair manifestations consistent with the diagnosis of PBT and several other maternal relatives had signs consistent with PBT. Particularly, only the probands manifested SNHL and severe intellectual disability, which have been reported rarely in piebald patients. The first descriptions of PBT associated with sensorineural deafness date back to the late sixties, however, these reports lack precise genetic data. Subsequently Spritz et al. [12] described a South African female affected by severe SNHL and PBT carrying a heterozygous missense change (p.R796G) in the KIT gene. Human homozygosity for KIT germline mutations has been reported in a severe multisystem phenotype consisting of hypopigmented skin and hair, blue irides, neurodevelopmental delay, hypotonia, SNHL, anemia, brachycephaly and clinodactyly [13,14]. In view of the uniqueness of the probands' phenotype and the rarity of neurological manifestations in PBT, we hypothesized the presence of other genetic changes.
In effect, we also found in both probands a heterozygous deletion at chromosome 15q13.3, overlapping to the 15q13.3 microdeletion syndrome minimal region (MIM #612001). This genomic disorder has been associated with variable neurological and behavioral symptoms, including cognitive impairment, epilepsy, deficits in social interaction, decreased attention spans, aggressive behaviors, autism, schizophrenia, and bipolar disorder [17][18][19]. Heterozygous deletion of chromosome 15q13.3 syndrome has incomplete penetrance and up to 75% of the described affected subjects inherited the variant from a parent with at least one of the neurodevelopmental or neuropsychiatric signs or an apparently normal phenotype [17][18][19]. The family history might be compatible with this syndrome because the deletion is likely inherited from the father, affected by severe schizophrenia. Interestingly, a paternal uncle was reported to be affected by depression.
As regards SNHL, it has never been described in association with the 15q13.3 microdeletion syndrome, despite its broad phenotypic spectrum. In conclusion, even though the presence of a third unidentified (probably autosomal recessive) genetic determinant cannot be excluded with certainty, our piebald probands could represent the second description of SNHL in carriers of a heterozygous germline mutation in KIT gene.

Conclusions
We describe a novel example of a complex disease in two twins, tracked back to at least two different autosomal dominant diseases which are maternally inherited PBT associated with KIT mutation and a 15q13.3 genomic disorder, likely inherited from the father, responsible for a neurodevelopmental disease with variable expressivity/incomplete penetrance.
To our best knowledge, this is the first reported phenotype due to the co-occurrence of germline pathogenic change in the KIT gene mutation and deletion at chromosome 15q13.3.
This report follows up a recent finding on large cohorts of complex patients showing 5% of cases having two or more genetic diseases. Our experience highlights the crucial role of genetic analyzes in the case of a multisystem phenotype, where the presence of multilocus syndromes should be considered [20,21]. Since two or more genetic disorders may overlap in a single individual, combining an extremely polymorphous multisystem phenotype, the diagnostic process should involve an interdisciplinary group of clinicians.
Funding: This research received no external funding.