Genetics of Hearing loss: from basic research to clinical applications

Background

Hearing loss (HL) affects one in every 1,000 births. The genetic and clinical heterogeneity of HL is vast. Since the introduction of molecular genetics, many advances have been achieved in elucidating the molecular basis of HL. Nevertheless, at least 60 HL loci still need the identification of the underlying gene. Moreover, mutations in more than 172 different genes have been reported in mice but only 44 of them linked to human HL. Thus, we still need to identify a large proportion of HL genes in humans. A more comprehensive description of the genetic networks underlying HL will improve the diagnosis and treatment of this common disease.

Aims

The project will focus on the forms of HL that are still un-elucidated from a genetic point of view applying "Nextgeneration" sequencing (NGS) to identify all or most forms. Whole Exome Sequencing (WES) will be used ensuring that most relevant genes will be discovered for a large cohort of Italian patients. Summarizing, the most relevant aims of the project are: a) Identification of additional genes involved in causing HL, including modifier genes, b) their validation in large population cohorts, c) definition of new pathways, d) accurate molecular epidemiology data to define diagnostic guidelines and recommendation, and to develop new diagnostic algorithms.

Study Design

During the last ten years we have analyzed more than 800 unrelated non-syndromic HL patients coming from all over Italy, and identified a group of 300 samples negative for the presence of a mutation in GJB2 and GJB6 genes as well as the A1555G mitochondrial mutation. Here, we propose to perform a comprehensive genetic variant discovery in these carefully phenotyped cases. To reach this goal we have planned the following strategy:

1. whole genome genotyping with high density SNPs array in all familial cases (despite their pedigree structure) to get some information to be used in the following filtering step (linkage);
2. WES followed by filtering the data tacking into account results obtained at the above mentioned step. Positive results will be confirmed by Sanger sequencing;
3. validation of new genes using in silico pathways analysis and whenever possible by immunohistochemistry in mice models;
4. definition of a molecular epidemiology picture by screening identified genes in a large cohort of cases by target resequencing (TS)

Summary Preliminary Data

A large cohort of 300 Italian HL cases, negative for the presence of GJB2, GJB6 and A1555G mitochondrial mutations has been already collected and is now available. The series includes cases with a variable degree of HL, ranging from mild to profound, and variable age of onset, from congenital to late onset. Moreover, several familial cases belonging to pedigree not large enough to be included in a linkage study but valuable for WES have been collected. WES is already in progress in a first series of 10 familial cases and some candidate genes are now under their final validation

Materials and methods

WES will be carried out coupling sequence capture on Agilent platform and sequencing on the Illumina or Solid platforms. After QC, data will be filtered using:

1. two databases (internal and dbSNPs)
2. linkage data if available.

Bioinformatics predictions about the putative effects of identified missense alterations will be made using differents tools (Mutatation Taster, Sift, Consurf, etc.). Summarizing, filtered bases will be annotated as follows:

1. novelty based on overlap with the SNP database, and an internal database;
2. effect on the encoded protein; 
3. evolutionary analysis to further help in elucidating their possible functional role using conservation information;
4. expression pattern using an internal database on genes expressed in the cochlea (inner ear).

Depending on the nature of identified disease gene(s), missense alterations will be tested by functional assays (e.g. mutations in ion channels/transporters/gap junction). Identified HL genes will be eventually further studied with respect to tissue localization by immunohistochemistry in mice models. Finally, in silico pathways will be generated using Ingenuity Software. After the analysis carried out as above described a given number of causative genes for HL will be identified, including genes already known to cause HL in humans. To define the molecular epidemiology of these genes we have planned a screening of 1000 Italian patients by TS. By this way, it would be possible to obtain accurate molecular epidemiology data, prerequisite for future testing protocols

Main Expected Results and Impact

This project will generate novel information on the causes of HL including new genes, new pathways, molecular epidemiology data, etc. Results will be the base for improving the diagnosis of HL by the definition of new diagnostics algorithms and by the development of accurate and reliable tools, for planning for more efficient prevention strategies, for making more accurate clinical predictions and classifications

Significance and Relevance for National Health System (SSN)

The absence of molecular epidemiology data as well as that on genotype-phenotype correlations makes that for many HL patients an accurate diagnosis and a precise counseling is still a dream. Moreover, the presence of a major gene (GJB2) has led to a growing and indiscriminate use of both prenatal and postnatal molecular analyses, leading, in most cases, to wrong and misleading information being delivered to patients and relatives. Knowledge arising from the project will lead to the availability of better diagnostic protocols providing:

1. more accurate insight in genotype-phenotype correlations,
2. define guidelines and recommendations on the basis of the molecular and clinical data,
3. the development of validated molecular diagnostic approaches and practical protocols,
4. reduction of the turn around times for molecular diagnostics.