WO2009080562A2 - Method and apparatus for detection of the healthy carrier state for the 35delg mutation in the gjb2 gene as risk factor for hearing loss - Google Patents

Abstract

A method is described for detection of the healthy carrier state for the genetic mutation 35delG in the GJB2 gene based on the measurement of epidermal thickness. Since this mutation correlates with the occurrence of genetically based hearing loss, the screening of a general normal hearing population for a potential genetic mutation, performed by non-invasive, rapid and reliable measurement of epidermal thickness, represents an important tool for genetic screening and assessment for a potential associated deafness risk. In addition to the method, the invention describes the apparatus to perform said method.

Description

Method and apparatus for detection of the healthy carrier state for the 35delG mutation in the GJB2 gene as risk factor for hearing loss Field of invention

The invention relates to a method for detection of the healthy carrier state for genetic mutations in the GJB2 gene based on the measurement of epidermal thickness. The detection of such condition can be correlated to the risk of onset of severe hearing loss in children where both parents have normal hearing but are carriers of a genetic mutation. Therefore, this method can be conveniently used to screen asymptomatic normal hearing individuals in order to select in the general population individuals at risk for developing hearing loss and thus candidates for further genetic diagnostic assessments. State of the art

Hearing loss affects about 4% of the population under the age of 45 years and includes a broad spectrum of clinical manifestations. In developed countries, at least 60-70% of cases of hearing loss is due to genetic causes, while the remainder is due to environmental causes, such as infections during pregnancy, trauma, drugs, etc. About 1/1000 births is affected by hypoacusia. About 30% of the genetic forms consist of syndromic conditions, among which, in terms of frequency, it is worth recalling the Alport's syndrome, Usher syndrome, Waardenburg syndrome, BOR (Branchio-Oto-Renal) syndrome, and the Pendred syndrome. The remaining 70% is characterized by non syndromic forms with Mendelian or mitochondrial inheritance, of which about 80% are transmitted as autosomal recessive trait, 18-20% are dominant, and the remainder are mitochondrial or X chromosome linked. After having ruled out a syndromic form, a possible genetic cause is searched. In this regard it should be reminded that the search for specific genes, involved in both syndromic and non syndromic forms, has so far led to the identification of over 1 10 loci associated with deafness but only 45 of the corresponding genes have been identified to date (see Hereditary hearing loss homepage at the address

Despite this considerable genetic diversity, reflecting at least in part a significant clinical heterogeneity, more than 60% of cases of non-syndromic deafness is due to mutations in genes that code for the proteins connexin 26 and connexin 30 (GJB2 and GJB6 genes, respectively). In particular, connexin 26 plays a key role in disease among white Caucasian population, with the presence of a very common mutation, named 35delG, which accounts for 70-80% of non-syndromic recessive forms. Healthy carriers who have normal hearing, and are identifiable only through invasive and expensive methods, occur in the general population at a rate that varies between 1/30 and 1/ 50.

Recently, a correlation between the GJB2 gene and epidermal thickness has been postulated, on the basis of literature data, only for carriers of the R143W GJB2 mutation that is typical of a tribe in Africa (Meyer CG, Amedofu GK, Brandner JM, Pohland D, Timmann C, Horstmann RD. Selection for deafness? Nature Med. 2002; 8(12):1332-3). In fact, direct bioptic measurement of epidermal thickness in a small number of mutation carriers and deaf revealed a significant increase of epidermal thickness compared to normal. Supporting a role of the R134W mutation, some experiments have been performed that show how keratinocytes, after insertion of this mutation, tend to form a thicker epidermis in an in vitro organotypic skin culture model. Furthermore, other authors have recently shown that cells expressing the R134W mutation are more sensitive than normal cells to the invasion by the enteric pathogen Shigella flexneri (Man YK, Trolove C, Tattersall D, Thomas AC, Papakonstantinopoulou A, Patel D, Scott C, Chong J, Jagger DJ, O'toole EA, Navsaria H, Curtis MA, Kelsell DP A Deafness-Associated Mutant Human Connexin 26 Improves the Epithelial Barrier In Vitro. J. Membr. Biol. 2007, 218(1 -3):29-37). This could explain the selective advantage of mutation carriers. To date, however, a correlation between the GJB2 gene and epidermal thickness has not been confirmed in different populations, nor has it been demonstrated for other mutations of this particular gene.

The above mentioned bioptic measurement of epidermal thickness employed in the African population, while giving accurate and reliable results, cannot be used for screening the general population in order to select a population at risk of hearing loss on a genetic basis because, in addition to being invasive, it is time consuming, requires experienced operators and, above all, would not be justified by the clinical condition of a subject without clear symptoms and/or signs of ongoing disease. Well known solutions for the measurement of tissue thickness involve the use of measurement systems based on both general and specialized echographic techniques (echographs), such as those used in the dermatological and ophthalmological fields. However, these solutions have clear drawbacks for the measurement procedure, due to the fact that a greater amount of information is produced by techniques for reconstruction of echographic images, or by specialized techniques focused on areas of interest for the field, that are not strictly coincident with and/or functional to the method we have found. Moreover, said non-specific general-purpose or specialized apparatuses involve high purchase, renting and operating costs, considerable encumbrance, weight and limited portability.

Moreover, well known solutions for measuring skin thickness involve mechanical measurements, sometimes not only by contact but also of invasive nature, characterized by lower precision and/or greater inconvenience or invasiveness for the patient.

Such solutions, however, have limited effectiveness because of their excessive costs, due to the fact that the first group makes use of instruments designed for more general applications and, in the second group, procedures are characterized by greater invasiveness. In addition, ultrasound systems are not very adaptable and flexible, especially when used for such measurements.

Known techniques are those of electrical, electromagnetic (for instance impedenziometric) nature, including optical techniques (visible and infrared) such as for instance tissue tomography, gas or liquid dynamic techniques and fluid dynamic techniques in general; such techniques are mostly experimental. Aims and summary

The present invention aims to provide a method for identification in a

Caucasian general population of potential healthy carriers of the 35delG genetic mutation in the GJB2 gene, a risk factor for hearing loss, in order to overcome inherent drawbacks present in the solutions according to known techniques referred above.

Therefore the object of the present invention is a method for detecting the state of potential healthy carrier of the genetic mutation 35delG in the GJB2 gene, as a risk factor for hearing loss, by means of ultrasonographic assessment of the epidermal thickness in normally hearing subjects, as in claims 1 to 7. The obtained measurement of epidermal thickness is compared with measurements of epidermal thickness in appropriate samples of the general population composed of normal subjects.

Further objects of the present invention are the apparatus for such evaluation and the computational method for calculation, as in claims 6 to 25, which is directly connected to a probe through interfaces and procedures that can be loaded in the memory of a computer and is capable of performing the steps of a procedure according to the invention when the calculation method is performed on a computer.

In addition to limiting the cost of the measurement system, the proposed solution allows the simple installation of a system that ensures the performance of measurements that are specific for the method of screening set forth. Brief description of the drawings

The invention will now be described, as an example without being limited thereto, with reference to the attached drawings, in which: figure 1 represents the architecture of an apparatus according to the invention; - figure 2 represents an example of the internal architecture of probes operating in the apparatus according to the invention; figures 2.1 and 2.2 represent simplified versions of the probe in fig. 2; figure 3 represents the internal architecture of the basic module operating in the apparatus according to the invention; - figure 4 shows the diagram of the operation of the apparatus according to the invention. Detailed description of the invention

Characteristics and advantages of the method and apparatus for assessment of the epidermal thickness as a parameter for detecting a possible 35delG genetic mutation in the GJB2 gene in normal hearing subjects, according to the invention, will be better understood from the following detailed description. The method of the invention is essentially a method for selecting a population at risk for developing hearing loss, on a genetic basis, linked to the genetic mutation 35delG in the GJB2 gene. The method is characterized by the fact that this mutation can be detected by ultrasonographic measurement of epidermal thickness in normal hearing subjects and by the fact that this measurement is compared with the measurement of epidermal thickness in an appropriate sample of the general population composed of normal subjects.

For the screening method for selecting a population at risk for developing hearing loss, on a genetic basis, linked to the genetic mutation 35delG in the GJB2 gene according the invention, the general population is preferably Caucasian and more preferably white Caucasian.

Therefore such method is for all intents and purposes a preliminary assessment of a given genetic susceptibility, correlating epidermal thickness with a possible genetic mutation in the GJB2 gene. However the relationship between epidermal thickness and GJB2 genetic mutation must be confirmed by specific gene analysis carried out on the DNA from selected subjects, performed by methods known to an expert in the field, such as DNA sequencing, PCR, etc. In fact, samples from subjects identified using the screening method of the invention must be further characterized for having true characteristics of healthy carrier of the genetic mutation 35delG in the GJB2 gene and the potential risk for hearing loss, demonstrating the presence of the genetic mutation by gene analysis on the DNA of the subject. Thus, in order to make the diagnosis of genetic mutation 35delG in the GJB2 gene, a further DNA analysis is necessary to define the GJB2 genotype of the subjects. In particular, the method for detecting a potential genetic mutation 35delG in the GJB2 gene, in normal hearing subjects, by ultrasonographic assessment of epidermal thickness, includes at least the following steps:

- ultrasound probe positioning orthogonally to the cutaneous plane of the repere point; - administering ultrasounds with relative and subsequent measurement of epidermal thickness at the level of the repere point; - calculating risk probability on the basis of a distinct linear regression curves for males and females and specific "cut-off" for male and/or female subjects: a) in the age group ranging from 20 to 80 years, and b) in reproductive age ranging from 20 to 49 years. The identification of the repere point is especially important for the method of the invention and preferably the ultrasonographic determination of epidermal thickness are carried out from the dermo-epidermal junction to the derma- hypodermal junction.

In fact, in a subject, the assessment of epidermal in different body districts shows minimal differences, which are negligible in terms of statistics, however, it clearly shows that the papillary dermis of the forehead, compared to other districts examined, is characterized by a hypoechoic band that is more evident than in the other districts studied, which better defines the transition between the epidermal germinative layer and the dermis. In addition, since the forehead area is not subject to wear, or covered with clothing that can exert an abrasive action, it provides the best basis for uniform sampling. These data, together with the ease of using the midline of the forehead as repere point and the easy accessibility even with a dressed, upstanding or, even better, seated subject, favored the choice of such district as the only skin area suitable for the evaluation. Moreover, it is extremely important that operators pay attention to keep the probe perpendicular to the skin plane, thereby avoiding oblique scans resulting in overestimated measurement of thickness. Materials that are well known to an expert in the field, for instance water or a gel, can be used to overcome the so- called "Fresnel dead zone" that is next to the transducer. In this regard, this objective is accomplished by interposition of a thick layer of gel (for example, about 5 ml) and by placing the transducer against the gel itself, without exerting any pressure.

When performing the measurement, it should be properly considered that what is actually measured is the so-called "entry echo" and not the actual thickness of the epidermal layer. In fact, the image that is obtained, consisting of a linear hyperechoic band well separated from the underlying dermis by a thin hypoechoic band, is the sum of the epidermal thickness and the leap of acoustic impedance of water or gel-epidermis. The thickness of the entry echo is thus higher than the actual epidermal thickness, however increased by a constant value. In fact, considering the intensity of the reflected echo as a relative constant that is proportional to the type of frequency used in accordance with the formula R = ( Z¹ - Z²)² / ( Z¹ + Z²)² with Z = r x v where Z = impedance and r = tissue density and v = velocity of ultrasound in the medium, we can say that the measurements made with probes of similar frequency are comparable, because the dependence of the rate on frequency (the dispersion) is less than 1 % within the range of frequencies commonly used for diagnosis. For the purpose of the invention, wide-band multifrequency linear probes can be used, for instance in the interval comprised between 6 and 50 MHz and beyond. In fact, comparative analyses between different probes, comprised within the above mentioned range (from 6 to 50MHz) has performed quite consistently with each other (Kendall concordance coefficient W>0.7 with significance p<0.05). Once the ultrasound measurement has been performed, it is possible to calculate risk probability. Linear regression curves, one for males and one for females, can be used in order to better discriminate between normal subjects and carriers of the genetic mutation. These linear regression analyses were obtained by stratifying the sample of this normal population by sex in the age group between 20 years and 80 years and using the programs for statistical calculation STATA9 and R2.5.1. The regression coefficients obtained from these tests were then used to obtain specific values (scores) of epidermal thickness directly adjusted by age and sex through the definition of two specific formulas (algorithms): one for males and one for females. The formula for males is: [thickness - (0.0082xage)-0.0961 ] while the formula for females is: [thickness - (0.0043x age)-0.0961 ]. With this type of formulas it is possible to use a cut-off of 0.16 both for males and females, enabling this test to identify 98.1 % of the carriers (sensitivity) with approximately 93% specificity. Always considering a cut-off of 0.16, the probability of a subject who tests positive (or the possibility for an individual with a value equal to or greater than 0.16) to be a real carrier is 14 times higher. Therefore, a subject randomly selected from the population who has a value equal to or greater than 0.16 has a 14-fold higher risk to be a carrier than a normal subject.

Considering that the test should be used for prenatal screening, a sub- analysis limited to the age group from 20 to 49 years was carried out. The upper limit has been identified on the basis of the definition of child-bearing age for women used by ISTAT, while for the lower limit it was decided to select the population over 20 years, given the limited number of subjects below this threshold (1 person in the group of carriers, no one in the group of healthy subjects). For this sub-analysis, an effort was made to identify a "cut-off" that was not-age-adjusted and expressed in millimeters, thereby was immediately usable without calculations. The analysis was conducted separately for males and females. For males, by setting the "cut-off" of the measurement at 0.75 mm (healthy subjects <0.75mm, carriers >=0.75mm), the test showed 100% sensitivity and 98.9% specificity, with positive and negative predictive values of 100% and 92.3%, respectively. Finally, positive and negative "likelihood ratios" are 91.0 and 0.0, respectively. For females, a "cut-off" of 0.64mm (healthy subjects <0.64mm, carriers >=0.64mm) provides a sensitivity and a specificity of 100.

The system, consisting of the apparatus and comprising also the appropriate computational method for detection of epidermal thickness, is essentially based on the use of a mobile ultrasound probe which can allow, by echo measurement techniques, an assessment of thickness of areas of the epidermis with the necessary precision, accuracy and speed.

The probe is configured to be activated only under conditions of proper placement when it operates in search mode, that is superimposed on a standard operating condition for acquisition and measurement. According to a further embodiment of the invention, the apparatus has a system architecture that provides for simplified management of the manual tasks for the operator, simplifying the pointing of the probe and relieving from operations of search and of optimal positioning for measurement, i.e it provides the possibility to detect the appropriate conditions for positioning and orthogonality. This simplifying feature, as it will be more detailed below, operates under both normal working and search conditions, allowing also an automatic transition between the two modes, once the signal of interest has been hooked. If desired, the apparatus can emit an acoustic signal related to the hooking event, in addition to the standard visual mode.

Figure 1 shows a schematic representation of a measuring apparatus according to the invention, indicated as a whole with reference 10. Reference 1 1 indicates multiple appropriate and compatible probes for echographic detection, which, however, have different or higher features and/or ergonomic aspects (shape, weight, smaller size); reference 12 indicates the basic module with an extractable data memory system (13) interfaced with a centralized data collection system (14). Such apparatus (10) with probes (1 1 ) and units (12, 13 and 14) is capable of running an application for acquisition of ultrasonic signals for data processing, to provide a measurement value with its own degree of uncertainty both in digital and graphic form, to run classification and diagnostic help algorithms and possibly to send acquired data and processed results to a remote computer or indicate a state of malfunction or incorrect use with appropriate sound or light signals.

A schematic example of ultrasound detection probe is shown in figure 2 as functional blocks, where reference 21 indicates the electrical/ultrasound transduction function, which carries an optical pointer system 22 on which are arranged a series of miniature angle transducers 23 or, alternatively, inclinometric transducers 24, at least one activation button 25, or even an indicator panel 26 or possible light and acoustic indicators, for testing the operating conditions and modalities, as well as a cable 27 for connection to the basic module 12.

The materials of choice are polymers for external medical use, the probe can be sterilized and, for reasons related both to adaptation of the acoustic impedance and to hygiene, will be covered with suitable caps, disposable septa or, in addition, can be covered with hydrogel and fluids currently used in the echographic field, taking into consideration also outpatient and hospital procedures in the field. Selected materials are also resistant to shocks and common solvents, while it is anticipated that specifications and labels will be provided in country-specific format, thereby avoiding specifications in a language other than the country of use, to promote the release, acceptance, adoption and comprehension by the various national markets. Shape and size of the measuring probe change based on the presence or absence of optional/accessory pointing modules, orthogonality and measurement indicator panels which are directly placed on the probe itself. The positioning and activation button 25 is placed in a site that prevents its accidental use and, at the same time, makes such button easy to find and comfortable to operate.

As indicated above, the apparatus allows the use of different types of probes. Those include a first model, known as pencil-like probe, which is more simple and less expensive and is similar to probes used for echography in the dermatological field, equipped only with the activation button, and a second model consisting in a special type of probe with a similar ultrasonic part but equipped with devices such as: a light pointing system, angle and inclination sensors and acoustic and light indicators of proper use/hooking, all of which are specific for the application and used by the device algorithm through automatic electronic identification of the model. The third type of probe contains the elements of novelty of the second one in addition to a data display module, in fact, this probe has the main features of the basic module and could act in an autonomous and wireless mode.

The set of probes 1 1 may then comprise also next generation probes containing a miniaturized processing module 30, allowing an even more practical use without the basic desk module and its connection cable.

Figure 3 shows a schematic view of the basic module 12, where in particular it is possible to see that probe 1 1 is connected and controlled through cable 27; it will be possible to place the module on a support suitable for the operations containing a printed circuit board in which is located a processing module 30 associated with a battery power supply 31. Furthermore, a panel for numerical and graphical representation of the data, equipped with buttons and switches 32 necessary for the operations, is placed in the frontal part of the support/container. The basic module 12 also comprises a battery charging circuit, one or more communication interfaces to remote host 34, a connector suitable to accomodate a removable data memory 35, and a reset button 36.

As it can be seen in Figure 3, the basic module 12 further comprises: - a microprocessor module 30, working as controller of the whole measurement system in all operating modes;

- a management software system, placed in part on the memory of the processing module 30 and in part (configuration and data) on the removable memory module 35.

The basic module 12 has firmware functionalities set in the processing module 30, which allow: a power management procedure to maintain the apparatus fully active in terms of measurement and rest (or stand-by) after a certain period of inactivity, for optimization of battery life; setting of measurement mode, search for the best positioning for measurement, hooking of the ultrasound signal, with the possibility to follow operator's manoeuvres and to inform that optimal conditions for acquisition have been achieved; - pre-processing of acquired data (management of masks, filtering, thresholds, correlations); remote management of configuration (firmware downloads, parameters, configurations, patient data).

Therefore, mobile probe 1 1 can be in the form of a pen according to ergonomic criteria functional to the scope of medical diagnostic application of the system.

Size, weight and shape of apparatus and probe are in line with the echographic characteristics of those for dedicated applications present on the market, for a greater acceptability by operators. The basic module 12 is expected to be of very simple use, small size and footprint and easy to be positioned/reclined on work tables or possibly on a cart for reading.

The basic module 12 of the apparatus represents the unit for control and management of the measurement. Such unit 12 configures and drives probe 1 1 , collects the signals coming from its transducers and, through an appropriate analog/digital conversion, performs the necessary algorithms and processing and therefore realizes the part of processed signals useful for a local representation on the display panel or a suitable formatting according to standard protocols for communication and interfacing that are present in computers currently available on the market.

The basic module 12 manages several information coming from the probes (ultrasonic, optic and angular) and provides an initial analysis in real time to verify the location and identify the echoes of interest and, depending on operator's maneuvers, will activate the appropriate processes for analysis.

In detail, the basic module 12 has the following features:

- connection through a single type of connector to multiple probes 1 1 , and in general to probes with diverse shapes and features, depending on their cost and available options, performing their identification by means of appropriate electrical signals present on the connector;

- processing group based on a microprocessor system integrated with the appropriate acquisition and data transmission interfaces; - local processing for display and measurement of data from probes 1 1 , processing of data coming from or directed to other peripheral devices, both at the level of optional/accessory features of probes 1 1 and of available features directed toward remote processing units, such as central processing systems, work stations, personal computers, portable computers, PDAs, mobile phones, entertainment stations and instruments suitable for storage, handling and printing of diagnostic data and related data.

From the point of view of internal architecture, the basic module 12 consists of a processor managed by an operating system suitable for "embedded" and portable systems. The basic module 12 is the core which enables the (interchangeable) probes 1 1 to operate and, at the end of the measurements, to put the results and the accessory data in the removable memory 13, or send via cable or wireless to remote units through interface 14. The software needed to manage the basic module 12 is configured to perform the following operations: - management of probes, their identification, management of sensors and actuators possibly present in them; - management of the protocol for measurement, data saving in the memory, recovery and update;

- management of communication protocol;

- management of the synchronization of information with remote computers;

- management of the push-button panel and graphic display screen towards the operator;

- integration with possible accessory devices, such as for instance local printing systems, external storage systems (hard disks, CD and DVD burners, systems for data transmission at a distance, even by means of mobile phones).

The measurement protocol implemented by the basic module 12 is now described.

The main objective of this protocol is to allow the operator to determine epidermal thickness in a simple and easy way.

The architecture must allow the creation of a record that is representative of the measurement, modalities, uncertainties and personal details of the study subject.

To that end, as mentioned, the basic module 12 preferably comprises a processor module (with micro-controller, permanent memory and data, interfaces for acquisition, for control of display monitor and keyboard, serial, USB, SD, ethernet), managed by a real-time operating system for embedded systems which is placed on a permanent memory, as well as the application procedure also permanently stored on memory. Through appropriate procedures for (even remote) maintenance and service, it will be possible to reprogram said permanent memories by rewriting with new versions of the operating system and of the application software.

Summarizing, the key features that must be implemented by the protocol are: - on the side of probes 1 1 : management of the ultrasonic sensor, of the optical pointer system, of the angle and inclination sensors, of piloting and signal conversion, as well as management of buttons and display systems present on the probe;

- on the side of the basic module 12: management of the buttons and display systems on the operating panel, removable memory management, management of the communication interface, of battery power and recharging;

- with regard to measurement: probe pointing, search for signs of echo and of optimal conditions, assessment of probe position relative to patient's epidermis via signals from angle and inclination transducers, hooking of the optimal echo signal by controlling frequency and amplitude, gain and multiple acquisitions, filtering and specific processing such as averages, correlations, transformations, running the algorithm for classification and assessment of the relationship with the reference values according to the classification of the patient under examination; - with regard to the removable memory module: management of widely spread and low cost memory modules such as: USB memory pens, memory modules for use in photography and mobile telephony, such as USB sticks, Compact Flash, SDs and the like; procedures for opening, writing, reading and for removal of the directory and folders used for data storage, as well as management of the configuration files of program memory and of the configuration parameters of the instrument;

- with regard to the communication part: management of a communication interface via cable, such as serial connections, USB, TCP-IP, Firewire, and the like, management of wireless communication through techniques and modules for radio connection (BlueTooth, Zig-Bee, WiFi, WiMax, etc.) or infrared (IrDa); management of the respective procedures for search, opening, closing the transmission session;

- management of system power supply, switch on, activation, instrument auto-shutdown, powering, recharging and charge status of batteries, as well as of the representation of the respective status signals, aiming at lower power consumption and maximum autonomy and availability of the instrument; - management of patient's data entry, of any operational mode, of configuration and maintenance data, both via the communication channels of module 34 and via user interface 32, 36 and the removable memory module 35; - management of self-testing and self-diagnostic procedures, both at switch-on and during running, of help procedures for use and maintenance even in electronic format in the system memory in addition to the usual paper format and instructions reported on the container. Such module works profitably in at least two configurations, including a standard configuration and a search/self-learning configuration, the transition from one to the other being based on pre-analysis of the data detected by module sensors. This occurs automatically and allows a simple and practical use of the system.

Furthermore the module is advantageously interfaced with one or more wireless computers through standard protocols which allow both an effective management of the measurements and the adoption of patient cards and relative databases, so it becomes easy to insert the features of the instrument even in a pre-existing hospital or outpatient data processing network, allowing data integration and management at the institutional level and the formulation of studies and clinical analyses. It should be noted that this allows also the connection to personal processing systems of doctors or researchers and the personal management of data archives, even remotely through digital cellular telephony.

According to another aspect of the invention, techniques are advantageously adopted for filing diagnostic and broadly clinical analyses for the purpose of activities such as screening, follow-up, etc..

Management and performance of the various features described above can be done, in whole or in part, by a computer program capable of implementing management functions for probe positioning, measurement of ultrasonic echoes and classification of the anatomical landmark (repere point) according to the regressions obtained from the search data.

More specifically, the computer program performs the following functions: management, positioning and hooking of probe signal through continuous correlation of ultrasound signals from angle and inclinometric sensors, mobile average of several measurements of epidermis thickness also as function of the two angles relative to the normal of patient's skin, search for significant data as compared to the series of previous measurements and management of the acoustic and optic hooking indicator; management of the measurement according to the previous point , along with a search for the mean across multiple points of measurement in order to avoid single measurements and search for the minimum average thickness of the epidermis, and relative determination of the confidence interval; running an algorithm for assignment of the measured skin thickness to the disease searched with assessment of the confidence interval of the assignment, according to the regression algorithm identified by the search and in relation to patient data (age, sex, ethnic group); running an algorithm for classification of the measurement that is not only linked to the regression parameters identified by the search, but is adaptive and based on measurements and classifications made during the use of the apparatus itself and on measurement, classification and regression data derived from knowledge developed and shared by the community of users of that apparatus, through self-learning techniques that are well known as knowledge database (KDB).

Therefore, the software can achieve the following steps, with reference to the flow chart in figure 4: activation and self-diagnosis; entry or reception from remote of patient data and parameters; search for echoes and hypoechoic areas to define the area of interest in the epidermis, deletion of spurious echoes, searches for hooking; - search for optimal measurement conditions (orthogonality) by hooking the reference angles; minimum measurement on lateral and longitudinal angles, and running an average of said measurements on more points, that are however all close, in order to avoid, by use of the average value, situations of single measurements; - tracking, measurement, value and error display; the data so obtained, combined with patient data, are subjected to a regression algorithm with appropriate analytical techniques with "cut-off" and clustering, to classify the patient group; sending data to remote computer and/or removable memory; - updating KDB parameters .

The computer program can be written using the C language, or ANSI-C that allows for easy migration.

Obviously, while maintaining the principle of the invention, the details of realization and the forms of implementation can be widely varied from what has been described and illustrated without for this going out of the scope of the present invention. In such perspective, it should be once more reminded that, even though the present description almost constantly refers to possible applications of the invention to a given context, for the sake of simplicity of illustration, the scope of the invention is general and therefore not limited to such specific operative/applicatory context.

Therefore, the method that is the object of the present invention can be profitably implemented in whole or in part by a computer program that includes media encoding for the realization of one or more steps of the method, when this program is run on a computer. Therefore, the scope of protection is meant to be extended to said computer program and also to computer-readable media that comprise a recorded message, said computer-readable media including media encoding a program for the realization of one or more steps of the method, when the program is run on a computer.

Experimental part The method for detecting the healthy carrier potential state of the genetic mutation 35delG in the GJB2 gene as risk factor for hearing loss by ultrasound determination of the thickness of the epidermis has been validated using three different types of commercial echotomographs: a Optikon HiScan device for ophthalmological use, equipped with single oscillating transducer in water bath S/N 39 with frequency of 35 MHz; - a multidisciplinary unit Esaote Mylab 25 with linear broadband probe, multifrequency LA 435 of 12 -17 MHz; a multidisciplinary unit Philips Envisor HD with linear broadband probe , multifrequency L 15-6 from 6 to 15 MHz.

While the device for ophthalmological use needs a system of contact with the skin surface consisting of a small cup filled with water, in which the transducer is immersed freehand, the probes of Esaote and Philips devices have a direct contact with the skin with the only interposition of a gel layer. Three different operators have studied 5 white Caucasian healthy volunteers, male, aged between

38 and 52 years, at the dorsal side of the forearm with the three types of probes; each operator repeated measurements of skin thickness on the same subject without being aware of the results obtained by other participants. The results obtained on epidermal thickness with three different operators showed the reproducibility of the data with the three different probes used (Kendall's coefficient of concordance W>0.7 with a significance of I p<0.05) (table 1 ). Table 1

Upon completion of the analysis described above, an extensive sample of normal white Caucasian subjects and of healthy white Caucasian carriers of mutations in the GJB2 gene was analyzed. The data obtained on epidermal thickness are shown in the following table 2 where subjects are divided by age group and sex.

Table 2

Deaf subjects homozygous for 35delG GJB2 mutation have been also analyzed. In homozygous patients a mean value of 0.98mm (± 0.14) was obtained for patients in the age comprised between 20 and 40 and of 1.06mm (± 0.08) in the group ranging from 40 to 60. This finding clearly demonstrates an even higher epidermal thickening process in patients as compared to carrier and normal controls.

The data obtained clearly support the role of the 35delG mutation of the GJB2 gene in determining a thickening of the epidermis in healthy carriers and also in deaf subjects (in a clear manner). It might open new perspectives in terms of early diagnosis/screening of hearing loss in combination with audiological screenings and followed by genetic tests.

In fact, in addition to the data obtained, running an ANCOVA analysis taking into account some variables such as age, sex, and GJB2 state (healthy or carrier), has shown that the three variables (age, sex, and GJB2 status) explain up to 75% of skin thickness. If the GJB2 status is removed from the analysis, the two remaining variables (age and sex) are not able to explain more than 26% of the variability of the skin thickness. This piece of data clearly shows an essential role of the GJB2 state (carrier of the 35delG mutation) in determining epidermal thickness.

Claims

Claims

1. A method for detecting in normal hearing subjects the state of potential healthy carrier of genetic mutation 35delG in the GJB2 gene by ultrasonographic measurement of epidermal thickness.

2. The method according to claim 1 comprising at least the steps of:

- positioning the ultrasonographic probe orthogonally to the cutaneous plane of the repere point;

- administering ultrasounds with relative and consequent measurement of the thickness of the epidermis at the level of the repere point; - calculating the risk probability based on a distinct linear regression curves for males and females and specific "cut-off" for male and/or female subjects: a) in the age group ranging from 20 to 80 years, and b ) in the age group ranging from 20 to 49 years.

3 The method according to claim 2, wherein the repere point is identified in the subject forehead.

4. The method according to claim 2, wherein the measurement of the epidermal thickness is performed with linear broadband multifrequency ultrasound probes in the interval comprised between 6 and 50 MHz.

5. The method according to claim 2, wherein the "cut-off" specific for subjects in the age group between 20 and 80 years calculated in comparison with normal subjects of the same age group is 0.16.

6. The method according to claim 2, wherein the "cut-off" specific for male subjects in the age group between 20 and 49 years calculated in comparison with normal subjects of the same age group is 0.75 mm.

7. The method according to claim 2, wherein the "cut-off" specific for female subjects in the age group between 20 and 49 years calculated in comparison with normal subjects of the same age group is 0.64 mm.

8. An apparatus for detecting in normal hearing subjects the state of healthy carrier for genetic mutations in the GJB2 gene by ultrasonographic assessment of epidermal thickness comprising:

- at least one ultrasonic probe module (1 1 ) which can be associated with at least one basic module, comprising one or more features (24, 25, 32, 33, 34) to detect and store said parameters, and an interfacing module (26) configured at least to transmit the data regarding said measurements (14) to other processing, storage and printing units;

- a device module (10) that comprises a microprocessor control module (30) configured to process parameters and to run the classification algorithm according to the results of said processing and to reference parameters present in the memory of said basic module (12) and to operate between at least two working conditions comprising:

- a first normal operating condition that comprises continuous measurements and evaluation of the data from position sensors in order to search the echoes of interest ;

- a second operating condition consists in hooking and accurate measurement controlled by said microprocessor module (30) if said results of said processing identify a stable data condition, comprising a phase of visual and acoustic signaling (12).

9. The apparatus according to the previous claim, wherein said at least one probe (1 1 ) and said at least one basic module (12) are configured to interface with removable memory modules according to standard management protocols.

10. The apparatus according to the previous claims, wherein said basic module (12) is configured to perform management functions of memory modules, said management functions comprising one or more of the following operations:

- opening archives and files in memory; - reading/writing and creation/deletion of files for measurement, diagnostics and configuration;

- file dating management;

- closing said files and archives.

1 1. The apparatus according to one or more of the previous claims, wherein said at least one probe (1 1 ) and said at least one basic module (12) are configured to interface with external processing, storage, printing systems according to standard protocols for their management, both in wired and wireless formats.

12. The apparatus according to one or more of the previous claims, wherein said basic module (12) is configured to perform management functions of communication protocols, such management functions comprising one or more of the following operations:

- opening of the dial-up connection;

- reading/writing and creation/deletion of files for measurement, diagnostics and configuration; - file dating management;

- closing said files and archives;

- closing the communication.

13. The apparatus according to one or more of the previous claims, wherein said apparatus (10) is configured to perform one or more of the following operations:

- identification of the type of probe connected to the basic module;

- management of angle sensors, probe inclination relative to patient's epidermis;

- assignment of a degree of quality to measurements in order to progress to the hooking and acquisition step;

- compare and classify the data with a database of references also according to patient's characteristics;

- releasing said measurements, performing their classification on graphic panels, memory and remote systems; - managing said self-learning data, updating the reference database;

- managing procedures of system self-diagnosis, as well as configuration parameters, maintenance and installation of their new versions and of new versions of the system software and applications.

14. The apparatus according to previous claims, wherein, in said previous operating conditions, said basic module provides for the management of recharging batteries and accumulators and the monitoring of the relative charge status.

15. The apparatus according to previous claims, wherein, in said previous operating conditions, said basic module provides for operations with simple echographic probes equipped only with the ultrasonic part and the button part, but without options, for least-costly versions.

16. The apparatus according to previous claims, wherein, in said previous operating conditions, said system provides for operating with ultrasonic probes equipped with laser pointer to facilitate the location of the epidermal area of interest.

17. The apparatus according to previous claims, wherein, in said previous operating conditions, said apparatus provides for operating with probes equipped with sensors of angle position and inclination relative to the surface of measurement.

18. The apparatus according to previous claims, wherein, in said previous operating conditions, said apparatus is expected to be made with miniaturization techniques that allow the housing of the features listed for the basic module directly inside the probe itself, featuring a more simple wireless format, without basic module, supports and connection cable.

19. The apparatus according to previous claims, wherein, in said previous operating conditions, said apparatus is expected to be made with minimum hardware techniques, possibly consisting only in the probe directly interfaced, without basic module, to external processing systems in which all processing features and the performance of the algorithm will be run by an application program to be installed in those external systems.

20. The apparatus according to previous claims, comprising connections to external processing/storage and printing systems using cable interconnection techniques.

21. The apparatus according to previous claims, comprising connections to external processing/storage and printing systems using wireless interconnection techniques.

22. The apparatus according to previous claims, comprising connections to removable memory modules.

23. The apparatus according to any of the claims 8 to 22, comprising the means for running software programs for managing the positioning of the probe, the measurement of ultrasonic echoes and the classification of the anatomical landmark according to regressions obtained from the search data.

24. The apparatus according to claim 23, wherein said means for running software programs comprise the means for running programs with software management capability, for positioning and hooking the probe signal through continuous correlation of ultrasound signals from angle and inclinometric sensors, mobile average of several measurements of epidermis thickness also as function of the two angles relative to the normal of patient's skin, search for significant data as compared to the series of previous measurements and management of the acoustic and optic hooking indicator.

25. The apparatus according to claim 24, wherein said means for running software programs perform the management of measurement with a search of the mean in several measurement points, in order to avoid single measurements and search for the minimum average thickness of the epidermis, and relative determination of the confidence interval.

26. The apparatus according to claim 23, wherein said means for running software programs comprise means for running an algorithm for assignment of the measured epidermal thickness to the disease searched with assessment of the confidence interval of the assignment, according to the regression algorithm identified by the search and in relation to patient data (age, sex, ethnic group).

27. The apparatus according to claim 23, wherein said means for running software programs comprise means for running an algorithm for classification of the measurement not only linked to the regression parameters identified by the search, but adaptive and based on measurements and classifications made during the use of the apparatus itself and on measurement, classification and regression data derived from knowledge developed and shared by the community of users of that apparatus, through self-learning techniques designated knowledge database (KDB).