Molecular Study Of Genetic Disorder

Published Date: 02 Nov 2017

The function and behavior of genes, the interrelationship between the information of macromolecules DNA and RNA and how heredity works are the key concerns of molecular genetics (Strachan, 2001). The goal of Geneticists is to understand how the information encoded in genes is transmitted from one generation to the next. They also analyze how little variations in genes can disrupt an organism’s development or cause abnormality.

Complete collection of an organism’s genetic material is called genome. The human genome consists of about 31,000 genes located on the 23 pairs of chromosomes in a human cell. Chromosomes occur in matched pairs within cell. Each chromosome has many genes, and each gene is located at a specific site on the chromosome, known as the locus. Like chromosomes, genes typically occur in pairs. A gene on one chromosome in a pair usually has the same locus as another gene in the other chromosome of the pair, and these two genes are called alleles. Alleles are the alternate form of the same gene.

How alleles work together to produce traits is described by patterns of inheritance (Bateson, 1902).The process of transmitting biological traits from parent to offspring through genes is called heredity. Mutations or alterations in DNA occur constantly. They create variations in the genes that are inherited. Mutation may benefit or harm an organism and results in the emergence of new types of genetic disorders.

2.2 DEAFNESS:

HUMAN EAR:

The human ear which performs dual function of balancing and perceiving sound is extremely complicated organ. The ear is situated in the temporal bone of the skull. The auditory system is made up of three discrete parts.

External Ear

Middle Ear

Inner Ear

2.3.1 THE EXTERNAL EAR:

It consists of Pinna and External auditory canal. The Pinna (or 'auricle') functions by of collecting more sound power and funnel it into the Auditory Canal, which is a cylindric tube 1cm across and 2.5 - 3 cm long. At the end of auditory canal is the Eardrum (or 'tympanic membrane') that completely closes it. The eardrum vibrates with the frequency of the sound by the sound waves funneled into the ear (Fig. I).

2.3.2 THE MIDDLE EAR:

The middle ear is the cavity about 1.3cm, connected with nose and throat by the Eustachian tube. It lies behind the eardrum; filled with the air, with a lining of mucous membrane. It has a chain of three ossicles (bones), named, the malleus, incus and stapes. The most lateral (toward the side of the head) of the three ear bones (ossicles) in the middle ear is called the malleus. The long process of the malleus (manubrium) is attached to the tympanic membrane. In response to sound tympanic membrane vibrates; making malleus to vibrates in synchronization. A series of two ossicles the incus, and the stapes is attached to the malleus, which in turn is attached via a foot plate to the oval window of the cochlea.

2.3.3 THE INNER EAR:

The inner ear consists of six mechano-receptive structures: three semicircular canals, utricle, saccule and the cochlea (Fig. II). The auditory system for hearing and the vestibular system for spatial orientation and equilibrium is simultaneously regulated by the inner ear. The auditory system specialized for detection of sounds is regulated by the Cochlea.

The inner ear consists of two parts:

A) The osseous or bony labyrinth and

B) The Membranous labyrinth

The osseous labyrinth is a series of cavities with the petrous portion of the temporal bone and membranous labyrinth is a series of communicating sacs and ducts which lie within the bony labyrinth (Hudspeth et al., 1989).

A) OSSEOUS LABYRINTH:

The osseous labyrinth is bony part of the inner ear. It is lined with the periosteum and is filled with the fluid known as perilymph having similar chemical composition as the cerebral spinal fluid. There are three semicircular canals, a cochlea and the vestibule. Cochlea consists of two and half turns in human, seen as a long coiled fluid filled tube resembling to a snail shell that is divided along its entire length by a partition. The two channels formed by the Cochlear partition are called the scala vestibule and the scala tympani respectively (Fig. IV) And they are also filled with perilymph. The length of cochlea is about 33mm in humans. The vibration has a different effect (resonance) at a different point along the basilar membrane depending on the frequency, accounting for passive tonotopy (Fig. III).

B) MEMBRANOUS LABYRINTH:

The membranous labyrinth consists of an elaborate series of communicating ducts and sacs immersed in the perilymph of the bony labyrinth. The portion of membranous labyrinth within the bony cochlea is known as scala media or the cochlear duct. The organ of corti which is the receptor organ of hearing lies within the scala media. Through a small tube the ductus reunions; the scala media joins the vestibular organ of vestibule, the saccule and utricle. The membranous labyrinth is continuous as the semicircular canals. Thus the six sensory structures of the inner ear i.e. three semicircular canals; utricle, saccule and the cochlea share the same continuous fluid environment, which is endolymph.

Endolymph differs from any other extracellular fluid found in the body, mainly compromises with K+ cation and very low in Na+. The source of K+ appears to be active transport by stria vascularis. On the other hand, the perilymph resembles in its chemical composition to the extra cellular fluid having high Na+ concentration. Since its osmolarity is similar to that of plasma, it is in osmotic equilibrium with blood (Graham, 2000).

2.3.4 THE ORGAN OF CORTI:

The receptor organ of hearing which is organ of corti lies within the scala media (Fig. IV). It is considered as the body's microphone. It rests on the basilar membrane, covered by tectorial membrane proteinaceous in nature. Auditory transduction is carried by the receptor cells called hair cells because of minute hair like microvilli (stereocilia) projecting from their apical surfaces. The stereocilia are arranged in V form on the surface of the hair cells in three rows and graded height. A small filamentous structure tip link extending from the upper end of each shorter stereocilium connects at a point above that level on the shaft of the nearest taller stereocilium. These tip links (Fig V) have main function in mechanoelectrical transduction process. The tallest stereocilia of outer hair cells directly contact the tectorial membrane.

2.3.5 HAIR CELLS:

The epithelial cells which originate from the surface of ectoderm not from the neural tube are called hair cells. Basically each hair cell is cylindrical or flask shaped. The hair cells are born upon other types of cells known as supporting cells. There are two kinds of hair cells on the basis of morphology and physiology, the outer hair cells (OHCs) and the inner hair cells (IHCs). The outer and inner hair cells of the organ of Corti change vibrational energy into neural energy that is transmitted via the auditory nerve to the brain (Fig. V).

OUTER HAIR CELLS (OHCS):

Usually there are 3-4 rows of outer hair cells in human ear. They receive only about 5% of the innervations of the nerve fibers from the acoustic portion of the VIII nerve although they are much greater in number than the inner hair cells. These cells contain muscle-like filaments that upon contraction stimulate and fine-tune the response of the basilar membrane to the movement of the traveling wave.

B) INNER HAIR CELLS (IHCS):

There is one row of approximately 3500 IHCs. These cells receive about 95% of the innervations from the nerve fibers from the acoustic portion of the VIII nerve. These cells have primary responsibility for producing our sensation of hearing. When lost or damaged, a severe to profound hearing loss usually occurs.

ear_diagram2

Figure: I Structure Of the Human Ear

b

Figure: II Structure of the Inner Ear

1. Anterior semicircular canal, 2. Ampulla (superior canal), 3. Ampulla (lateral canal), . Sacculus, 5. Cochlear duct, 6. Helicotrema, 7. Lateral (horizontal) canal, 8. Posterior canal

9. Ampulla (posterior canal), 10. Oval window, 11. Round window, 12. Vestibular duct (scala vestibuli), 13. Tympanic duct (scala tympani), 14. Utricule

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Figure: III Frequency Distribution along the Human Chochlea Basilar Membrane (Passive Tonotopy)

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Figure: IV Overall Anatomy of Human Ear

Figure: V Structure of the Hair Cell

2.4 MECHANISM OF NORMAL HEARING:

The hearing process consists of three parts:

Conductive

Sensory

Neural

2.4.1CONDUCTIVE:

Conductive hearing begins with the auricle capturing sound and channeling it into the external auditory canal, which leads to the tympanic membrane. In the first part of the hearing process, sound waves cause the tympanic membrane to vibrate. These vibrations are then conducted through the three bones of the middle ear. As the tympanic membrane moves, it causes the malleus to vibrate, which in turn vibrates the incus and then the stapes. The stapes moves against the window, transmitting vibrations to the fluid in the cochlea of the inner ear.

2.4.2SENSORY:

In the second part of the hearing process, pressure waves of cochlear fluid are transferred into the spiral-shaped cochlea. Vibrations from the movement of fluid displace the basilar membrane in the organ of Corti. The displacement is directly proportional to loudness of sound. The resultant movement displaces the hairs attached to the hair cells, causing depolarization (change in electrical charge from positive to negative) of the hair cells (Fig. VI&VII). The organ of Corti converts the mechanical energy of the vibrating fluid into electrical energy in this way. The basilar membrane varies in width and tension to detect different sounds. Bearing in mind that the cochlea is like a snail shell, high frequency sounds are detected near the oval window (the opening of the shell), and low frequency sounds are detected near the apex of the cochlea (deep inside the spiral core of the shell) (Fig. III).

2.4.3NEURAL:

The depolarized hair cells create an electrical impulse in the cochlear nerve in the third part of the hearing process. These nerve impulses are sent through the cochlear nerve to the brain (cerebral cortex of the opposite temporal bone) where they are processed as sound. Different areas of the primary auditory complex of the brain are excited by sounds of different frequencies (Fig. IX). The cells are thought to recover from the stimulus by pumping out the potassium through gap junctions (Connexin channels) and voltage gated potassium channels (Petit et al., 1996) (Fig. V).

Hearing loss can result if any pathway in this chain is disturbed, either by birth or later on in life, due to some injury or drug usage. High proportions of hearing loss cases are due to outer hair cell abnormalities(Kossal et al., 1997).

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Figure: VI Shearing Of Hair Cells and Production of Action Potential

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Figure: VII Role of Sterocilia in Production of Action Potential

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Figure: VII Functional Polarization of Hair Cells

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Figure: IX Tonotopic Organization in Primary Auditory Cortex of the Brain

2.5CLASSIFICATION:

Hearing loss is classified by type, onset, severity, frequency, and cause and inheritance pattern.

2.5.1TYPE:

Conductive hearing loss results from abnormalities of the external ear and/or the ossicles of the middle ear. In contrast, sensorineural hearing loss results from the malfunction of inner ear structures such as the cochlea. Mixed hearing loss is a combination of both conductive and sensorineural hearing loss, and central auditory dysfunction results from damage or dysfunction at the level of the eighth cranial nerve, auditory brainstem, or cerebral cortex (Fig X).

Figure: X Classification on basis of Type

2.5.2ONSET:

Prelingual hearing loss is present before speech develops. All congenital hearing loss is prelingual, but not all prelingual hearing loss is congenital. Postlingual hearing loss occurs after the development of normal speech (Fig XI).

Figure: XI Classification on basis of Onset

2.5.3SEVERITY:

Hearing is measured in decibels (dB). The threshold or 0 dB mark for each frequency refers to the level at which normal young adults perceive a tone burst 50% of the time. Hearing is considered normal if an individual’s thresholds are within 15 dB of normal thresholds. To calculate the percent hearing impairment, 25 dB is subtracted from the pure tone average of 500 Hz, 1000 Hz, 2000 Hz, and 3000 Hz. The result is multiplied by 1.5 to obtain an ear-specific level. Impairment is determined by weighting the better ear five times the poorer ear (Fig XII).

Note:

Because conversational speech is at approximately 50 to 60 dB hearing level (HL), calculating functional impairment on the basis of pure tone averages can be misleading. For example, a 45 dB hearing loss is functionally much more significant than 30% implies (Northern , et al 2002).

A different rating scale is appropriate for young children, for whom even limited hearing loss can have a great impact on language development (Northern, et al 2002).

Figure: XII Classification on basis of Severity

2.5.4FREQUENCY:

On the basis of frequency hearing loss is classified as Fig XIII.

Figure: XIII Classification on basis of Frequency

2.6 EPIDEMIOLOGY OF HEARING LOSS

2.6.1PREVALENCE AND CAUSE:

Profound hearing loss is present in 1 of every 1000 to 2000 newborn infants. However, milder but still clinically significant degrees of unilateral or bilateral hearing loss occur in an additional one to two infants (Morton, 1991; White, 2003). More than 50% of all prelingual deafness cases are hereditary in nature, with the remaining 40% to 50% of cases secondary to environmental factors such as infectious or iatrogenic causes (Fig. XIV) (Marazita, et al 1993; Cohen, et al 1995). The majority of genetic hearing loss diagnosed in infancy and early childhood is autosomal recessive in inheritance and nonsyndromic. Generally, the prevalence of hearing loss increases with age, reflecting the important impact of both genetics and the environment in its development. Indeed, interactions between environmental triggers and an individual’s genotype can contribute to the development of hearing loss. For example, aminoglycoside induced toxicity is potentiated on a genetic background that includes the mitochondrial DNA 1555 A-to-G mutation (Fischel, 1999).

2.6.2ENVOIRMENTAL CAUSES OF HEARING LOSS:

prenatal infections from "TORCH" organisms (i.e., toxoplasmosis, rubella, cytomegalic virus, and herpes), or postnatal infections, particularly bacterial meningitis caused by Neisseria meningitidis, Haemophilus influenzae, or Streptococcus pneumonia in children commonly results in Acquired (environmental) hearing loss. Meningitis from many other organisms, including Escherichia coli, Listeria monocytogenes, Streptococcus agalactiae, and Enterobacter cloacae, can also be responsible for hearing loss. Association with asymptomatic congenital cytomegalovirus (CMV) infection is often unrecognized and can be cause of variable, fluctuating, sensorineural hearing loss.

Acquired hearing loss in adults is usually caused by environmental factors, especially noise exposure, but susceptibility probably reflects an environmental-genetic interaction. For example, an A-to-G transition at nucleotide position 1555 in the mitochondrial genome (mtDNA) results in aminoglycoside-induced hearing loss (Kalatzis & Petit et al, 1998). Age-related hearing loss (presbycusis) is acquired and characterized by diminished hearing sensitivity and speech comprehension in noisy environments, slowed central processing of acoustic information, and impaired localization of sound. Affected persons may have difficulty in conversation, music appreciation, orientation to alarms, and participation in social activities (Gates and Mills, 2005).

Fig: XIV Hereditary and environmental causes of congenital hearing loss.

2.6.3HEREDITARY CAUSES OF HEARING LOSS:

2.6.3.1SYNDROMIC HEARING IMPAIRMENT:

Syndromic hearing impairment accounts for up to 30% of prelingual deafness, but its relative contribution to all deafness is smaller because of the impact of postlingual hearing loss.

AUTOSOMAL DOMINANT SYNDROMIC HEARING IMPAIRMENT:

A) WAARDENBURG SYNDROME:

Waardenburg syndrome (WS) is the most common type of autosomal dominant syndromic hearing loss. It consists of variable degrees of prelingual nonprogressive sensorineural hearing loss accompanied by pigmentary abnormalities of the skin, hair (white forelock), and eyes (heterochromia iridis). Because affected persons may dye their hair, the presence of a white forelock should be specifically sought in the history and physical examination. Four types are recognized—WS I, WS II, WS III, and WS IV—based on the presence or absence of other abnormalities.WS I and WS II share many features but have an important phenotypic difference; WS I is characterized by the presence of a lateral displacement of the inner canthus of the eye, which is known as dystopia canthorum, whereas in WS II this feature is absent. In WS III upper-limb abnormalities are present, and in WS IV, Hirschsprung disease is present. Mutations in PAX3 cause both WS I and WS III, and mutations in MITF and SNA12 are responsible for some cases of WS II (Baldwin et al, 1999; Tassabehji et al, 1992; Hoth et al, 1993; Sa´nchez et al, 2002). Mutations in EDNRB, EDN3, and SOX10 have been confirmed to cause WS IV (Edery et al, 1996; Hofstra et al, 1996; Pingault et al, 1998).

B) BRANCHIO-OTO-RENAL SYNDROME:

Branchio-Oto-Renal (BOR) syndrome is the second most common type of autosomal dominant syndromic hearing loss. The BOR phenotype includes conductive, sensorineural, or mixed hearing loss; branchial cleft cysts or fistulae; malformations of the external ear; preauricular pits; and renal anomalies (Melnick et al, 1975; Fraser et al, 1978). Penetrance is high, and expressivity is extremely variable. In approximately 40% of families segregating a BOR phenotype, mutations in the EYA1 gene have been identified. Mutations in SIX1 were recently discovered in a few BOR families without EYA1mutations (Ruf et al, 2004), which is consistent with the known interaction of EYA1 and SIX1 proteins in transcriptional regulation and their involvement in the development of the mammalian ear and kidney(Kalatzis et al, 1998; Xu et al, 2003; Zheng et al, 2003). There are a large number of BOR cases that appear to be genetic but for which no mutation has been identified.

C) STICKLER SYNDROME:

Stickler syndrome is an autosomal dominant disorder of type 2 collagen resulting in sensorineural hearing loss, cleft palate, congenital myopia, and spondyloepiphyseal dysplasia that eventually leads to osteoarthritis.Thesyndrome iscommon,and three forms are recognized based on the precise molecular genetic defect involved. STL1 is caused by mutations in COL2A1, STL2 is caused by mutations in COL11A1, and STL3 occur secondary to mutations in COL11A2 (Williams et al, 1996; Richards et al, 1996; Vikkula et al, 1995; Wilkin et al, 1998). STL1 and STL2 are characterized by severe myopia, which predisposes individuals to retinal detachment. Myopia is absent in STL3 because the COL11A2 gene is not expressed in the eye. Because of the substantial risk for retinal detachment, ophthalmologic assessment is mandatory in all persons diagnosed with STL1 and STL2.

D) NEUROFIBROMATOSIS 2:

Neurofibromatosis 2 (NF2) is associated with a rare, potentially treatable type of deafness. The hallmark of NF2 is hearing loss secondary to bilateral vestibular schwannomas. Impairment primarily begins in the third decade, concomitant with the growth of a vestibular schwannoma, and is generally unilateral and gradual but may be bilateral and sudden (Yohay, 2006). A retrocochlear lesion can often be diagnosed by audiologic evaluation, although the definitive diagnosis requires magnetic resonance imaging with gadolinium contrast. Affected persons are at risk for a variety of other tumors including meningiomas, astrocytomas, ependymomas, andmeningioangiomatosis. Mutations in NF2, which encodes the protein Merlin, are causative, and molecular genetic testing of NF2 is available for pre symptomatic, at-risk family members to facilitate early diagnosis and treatment.

AUTOSOMAL RECESSIVE SYNDROMIC HEARING IMPAIRMENT:

A) PENDRED SYNDROME:

Pendred syndrome is the most common form of syndromic hearing loss and is characterized by congenital severe-to-profound sensorineural hearing impairment, structural defects of the temporal bone and inner ear, and euthyroid goiter. Goiter is not present at birth and develops in early puberty (40%) or adulthood (60%). Delayed organification of iodine by the thyroid can be documented by a perchlorate discharge test. The hearing loss is associated with an abnormality of the bony labyrinth (Mondini dysplasia or dilated vestibular aqueduct) that can be diagnosed by computed tomography of the temporal bones (Phelps et al, 1998; Reardon et al, 2000). Vestibular function is abnormal in the majority of affected persons. Mutations in SLC26A4 (PDS) can be identified in approximately 50% of multiplex families with a Pendred syndrome phenotype, and genetic testing of this gene is appropriate for persons with Mondini dysplasia or an enlarged vestibular aqueduct and progressive hearing loss. Early studies reported that Pendred syndrome accounted for up to 7.5% of congenital deafness, but contemporary studies suggest that the prevalence of Pendred syndrome is lower (Newton, 1985; Arnos et al, 1992). Mutations in SLC26A4 also cause autosomal recessive nonsyndromic hearing loss at the DFNB4 locus.

B) USHER SYNDROME:

Usher syndrome is one of the most common types of autosomalvrecessive syndromic hearing loss and is characterized byvdual sensory impairments. Affected individuals are born withvsensorineural hearing loss and early in life develop retinitis pigmentosa, a progressive degeneration of the retina that leadsvto loss of night vision, restriction of visual fields, and blindness by adolescence. It is of significance to note that the visual impairment from retinitis pigmentosa is usually not apparent in the first decade, thus making funduscopic examination before 10 years of age of limited utility. However, electroretinography can identify abnormalities in photoreceptor function in children as young as 2 to 4 years of age. During the second decade, night blindness and loss of peripheral vision become evident and inexorably progress.

Usher syndrome is both phenotypically and genotypically heterogeneous. Three types are recognized on the basis of the degree of hearing impairment and vestibular function. Usher syndrome type I is characterized by congenital severe-to-profound sensorineural hearing loss and vestibular dysfunction. Affected persons find traditional sound amplification ineffective and usually communicate manually. Because of the vestibular deficit, developmental motor milestones for sitting and walking are generally achieved later than normal. Usher syndrome type II is characterized by congenital mild-to-severe sensorineural hearing loss and normal vestibular function. Hearing aids provide effective sound amplification for these persons, and their communication is usually oral. Usher syndrome type III is the rarest form and is characterized by progressive hearing loss and progressive deterioration of vestibular function (Kimberling et al, 1989).

C) JERVELL AND LANGE-NIELSEN SYNDROME:

Jervell and Lange-Nielsen syndrome is the third most common type of autosomal syndromic hearing loss. The syndrome consists of congenital sensorineural hearing loss and prolongation of the QT interval as detected by electrocardiography (the abnormal QTc [corrected] is_440 ms). Affected individuals have syncopal episodes and may have sudden death. Although a screening electrocardiogram is not highly sensitive, it may be suitable for screening deaf children. High-risk children (i.e., those with a family history that is positive for sudden death, sudden infant death syndrome, syncopal episodes, or long QT syndrome) should have a thorough cardiac evaluation. Mutations in two genes, KCNQ1 and KCNE1, have been identified in affected persons (Neyroud et al, 1997; Schulze et al, 1997).

D) BIOTINIDASE DEFICIENCY:

Biotinidase deficiency is secondary to absence of the watersoluble B-complex vitamin biotin. Biotin covalently binds to four carboxylases that are essential for gluconeogenesis, fatty acid synthesis, and catabolism of several branched-chain amino acids. If biotinidase deficiency is not recognized and corrected by daily addition of biotin to the diet, affected persons develop neurologic features such as seizures, hypertonia, developmental delay, ataxia, and visual problems. Furthermore, in at least 75% of children who become symptomatic, sensorineural hearing loss develops and can be both profound and persistent even after treatment is initiated (Wolf et al, 2002). Cutaneous features are also present and include a skin rash, alopecia, and conjunctivitis. With treatment that consists of biotin replacement, the neurologic and cutaneous manifestations resolve; however, the hearing loss and optic atrophy are usually irreversible. Therefore, if a child presents with episodic or progressive ataxia and progressive sensorineural deafness, with or without neurologic or cutaneous symptoms, biotinidase deficiency should be considered. To prevent metabolic coma, diet and treatment should be initiated as soon as possible (Wolf et al, 2003; Heller et al, 2002).

E) REFSUM DISEASE:

Refsum disease is a postlingual severe progressive sensorineural hearing loss associated with retinitis pigmentosa, peripheral neuropathy, cerebellar ataxia, and elevated protein levels in the cerebrospinal fluid without an increase in the number of cells (Wander et al 1995). It is caused by defective phytanic acid metabolism, and as such, the diagnosis is established by determining the serum concentration of phytanic acid. Two genes, PHYH and PEX7, have been implicated in the majority of Refsum cases, although a small number of patients exist in whom mutations have not been found (van den Brink and Wanders, 2006). Although Refsum disease is rare, it is important that it be considered in the evaluation of a deaf person because it can be easily treated with dietary modification and plasmapheresis.

X-LINKED SYNDROMIC HEARING IMPAIRMENT:

A) ALPORT SYNDROME:

Alport syndrome is characterized by progressive postlingual sensorineural hearing loss of varying severity, progressive glomerulonephritis leading to end-stage renal disease, and variable ophthalmologic findings such as anterior lenticonus. Hearing loss usually does not manifest before the first decade of life, and the progressive myopia secondary to anterior lenticonus is considered by some authors to be sufficient to diagnose Alport syndrome (Govan, 1983). Autosomal dominant, autosomal recessive, and X-linked inheritance are described. X-linked forms account for approximately 85% of cases and are attributable to mutations in COL4A5, a member of the type IV collagen gene family (Barker et al, 1990). Autosomal recessive inheritance accounts for the majority of the remaining 15% of cases, with only a few autosomal dominant cases documented.

B) MOHR-TRANEBJAERG SYNDROME (deafness-dystonia-optic atrophy syndrome)

Mohr-Tranebjaerg syndrome was first described in a large Norwegian family with apparent progressive postlingual nonsyndromic hearing impairment and classified as DFN1 (Mohr and Mageroy, 1960). Reevaluation of this family, however, revealed additional findings, including visual disability, dystonia, fractures, and mental retardation, indicates that this form of hearing impairment is syndromic rather than non syndromic (Tranebjaerg et al, 1992). The gene for this syndrome, TIMM8A, is involved in the translocation of proteins from the cytosol across the inner mitochondrial membrane (TIM system) and into the mitochondrial matrix (Jin et al, 1996; Koehler et al, 1999).

MITOCHONRIAL SYNDROMIC HEARING IMPAIRMENT:

Mitochondrial DNA mutations have been implicated in a variety of diseases ranging from rare neuromuscular syndromes, such as Kearns-Sayre syndrome; mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes; myoclonic epilepsy and ragged red fibers; and neuropathy, ataxia, and retinitis pigmentosa syndrome; to common conditions, such as diabetes mellitus, Parkinson disease, and Alzheimer disease. One mutation, the 3243 A-to-G transition in the gene MTTL1, has been found in 2% to 6% of individuals with diabetes mellitus in Japan (Usami et al, 2000). Sixty-one percent of patients with diabetes mellitus carrying this mutation also have hearing loss (Majamaa et al, 1998; Kadowaki et al, 1994). The hearing loss is sensorineural and develops only after the onset of the diabetes mellitus. The same mutation is associated with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes, raising questions of penetrance and tissue specificity, issues further confounded by heteroplasmy associated with mitochondrial conditions.

2.6.3.2NONSYNDROMIC HEARING IMPAIRMENT:

More than 70% of hereditary hearing loss is non syndromic (Cremers et al, 1991; Van Camp et al, 1997). Disorders discussed in this section are organized by mode of inheritance.The different gene loci for non syndromic deafness are designated DFN (for DeaFNess). Loci for genes inherited in an autosomal dominant manner are referred to as DFNA, loci for genes inherited in an autosomal recessive manner are referred to as DFNB, and loci for genes inherited in an X-linked manner are referred to as DFN. The number following these designations reflects the order of gene mapping and/or discovery. It is important to realize that several recessive and dominant loci have been mapped to the same chromosomal region, and in many of these cases, allelic variants of a single gene have been found. Examples include DFNB2 and DFNA11, both of which map to 11q13.5 and are caused by mutations in MYO7A, the gene that also causes Usher syndrome 1B, and DFNB21 and DFNA8/12, both of which are caused by mutations in TECTA.

A) AUTOSOMAL DOMINANT NONSYNDROMIC HEARING IMPAIRMENT:

The characteristic phenotype of a person with autosomal dominant nonsyndromic hearing impairment is a progressive postlingual hearing loss that begins in the second to third decades of life. The condition is extremely heterogeneous, with multiple genes implicated in its pathogenesis. Audioprofiles may be distinct and therefore useful in predicting candidate genes for mutation screening. For example, mutations in WFS1 are found in 75% of families segregating autosomal dominant nonsyndromic hearing impairment that initially affects the low frequencies while sparing the high frequencies.

B) NON-SYNDROMIC AUTOSOMAL RECESSIVE DEAFNESS:

Various mapped loci for non-syndromic autosomal recessive hearing loss are symbolized as DFNB1, DFNB2 and so on in the order in which they are first reported or reserved. To date 95 non-syndromic recessive deafness loci have been mapped and twenty defective genes have been identified encoding connexin-26 (GJB2) at DFNB1 locus (Kelsell et al., 1997), Myosin VIIa (MYO7A) at DFNB2 locus (Liu et al., 1997), Myosin XV (MYO15) at DFNB3 locus (Wang et al., 1998), Pendrin (SLC26A4) at DFNB4 locus (Li et al., 1998), TMIE at DFNB6 locus (Naz et al., 2002), Tranmembrane choclear expressed gene1 (TMC1) at DFNB7/11 and DFNA36 (Kurima et al., 2002) Otoferlin (OTOF) at DFNB9 locus (Chaib et al., 1996a), Transmembrane serine protease-3 (TMPRSS3) at DFNB8/10 locus (Bonnetamir et al., 1996), Cadherin 23 (CDH23) at DFNB12/USHER1D locus (Chaib et al., 1996b), STRC at DFNB16 locus (Verpy et al., 2001), Harmonin (USH1C) at DFNB18/USHER1C locus, α-Tectorin (TECTA) at DFNB21 locus (Mustapha et al., 1999), Otoancorin (OTOA) at DFNB22 locus (Zwaenepoel et al., 2002), Protocadherin 15 (PCDH15) at DFNB23/USHER1F (Ahmed et al., 2003), RDX at DFNB24 locus (Khan et al., 2007),TRIOBP at DFNB28(Riazuddin et al.,2006), Claudin-14 (CLDN14) at DFNB29 locus (Wilcox et al., 2001), MYO6A at DFNB37 locus (Ahmad et al., 2003), MYO3A at DFNB30 locus (Walsh et al., 2002) and Whirlin (WHRN) at DFNB 31 locus (Mburu et al., 2003), ESPN at DFNB36 locus (Naz et al., 2004), TRIC at DFNB 49 locus (Riazuddin et al., 2006), COL11A2 at DFNB53 locus (Chen et al.,2005), PJVK at DFNB59 locus (Delmaghani et al., 2006) LHFPL5 at DFNB66/67 locus (Shabbir et al.,2006), MRSRB3 at DFNB74 locus (Waryah et al., 2009 ; Ahmed et al., 2011 ) LOXHD1 at DFNB77 (Grillet et al., 2009), TPRN at DFNB79 (Rehman et al., 2010 ; Li et al., 2010) ,GPSM2 at DFNB82(Walsh et al., 2010), PTPRQ at DFNB84(Schraders et al., 2010), GJB3 at DFNB91(Liu et al., 2000) and SERPINB6 (Sirmaci et al., 2010)(Fig XV) (Table 2.1).

All these loci result in hearing loss and so far no other clinical features are associated with them with the exception of DFNB4, which is associated with enlarged vestibular aqueduct (Li et al., 1998). DFNB2, DFNB4 and DFNB21 gene identification was aided as they had positional candidates because other syndromic or dominant deafness loci had previously mapped to the same chromosomal locations and disease causing genes identified.

DEAFNESS MAP LOCATIONS

Fig: XV Cytogenetic map positions of human nonsyndromic deafness loci (modified from Friedman and Griffith, 2003). A deafness locus is underlined when the gene is known. Loci with published, statistically significant support for linkage are shown with a solid black font. Shown with a gray font are loci for which there are reserved symbols but no published data, or published loci lacking statistically significant support for linkage. DFN is the root of the locus symbol for deafness. An A or B suffix indicates that the mutant allele is segregating as an autosomal dominant or autosomal recessive, respectively. Sex-linked nonsyndromic hearing loss is designated with a DFN symbol and a numerical suffix. DFNM1 on chromosome 1q24 is a dominant modifier of DFNB26 on chromosome 4q31.

Table: 2.1 Loci for nonsyndromic autosomal recessive deafness

Locus (OMIM)

Location

Gene (OMIM)

Important reference

DFNB1

13q12

GJB2

Guilford et al., 1994 ; Kelsell et al., 1997

DFNB2

11q13.5

MYO7A

Guilford et al., 1994 ; Liu et al., 1997 ; Weil et al., 1997

DFNB3

17p11.2

MYO15A

Friedman et al., 1995 ; Wang et al., 1998

DFNB4

7q31

SLC26A4

Baldwin et al., 1995 ; Li et al., 1998

DFNB5

14q12

unknown

Fukushima et al., 1995

DFNB6

3p14-p21

TMIE

Fukushima et al., 1995 ; Naz et al, 2002

DFNB7/11

9q13-q21

TMC1

Jain et al., 1995 ; Scott et al., 1996 ; Kurima et al., 2002

DFNB8/10

21q22

TMPRSS3

Veske et al., 1996  ;  Bonné-Tamir et al., 1996  ;  Scott et al., 2001  

DFNB9 

2p22-p23

OTOF

Chaib et al., 1996 ;  Yasunaga et al., 1999

DFNB10

see DFNB8   

 

 

DFNB11

see DFNB7

 

 

DFNB12

10q21-q22

CDH23

Chaib et al., 1996 ;  Bork et al., 2001 

DFNB13

7q34-36

unknown

Mustapha et al., 1998

DFNB14

7q31

unknown

Mustapha et al., 1998

DFNB15

3q21-q25

19p13

 

GIPC3  

Chen et al., 1997

Charizopoulou et al., 2011

DFNB16

15q21-q22

STRC

Campbell et al., 1997 ; Verpy et al., 2001

DFNB17

7q31

unknown

Greinwald et al., 1998

DFNB18

11p14-15.1

USH1C

Jain et al., 1998  ; Ouyang et al., 2002 ; Ahmed et al., 2002

DFNB19

18p11

unknown

The Molecular Biology of Hearing and Deafness meeting Bethesda, October 8-11, 1998 (Green et al., abstract 108) 

DFNB20

11q25-qter

unknown

Moynihan et al., 1999

DFNB21

11q

TECTA

Mustapha et al., 1999

DFNB22

16p12.2

OTOA

Zwaenepoel et al ., 2002

DFNB23

10p11.2-q21

PCDH15

Ahmed et al, 2003

DFNB24

11q23

RDX

Khan et al., 2007

DFNB25

4p13

GRXCR1  

Schraders et al., 2010  

DFNB26 

4q31

unknown

Riazuddin et al., 2000

DFNB27

2q23-q31

unknown

Pulleyn et al., 2000

DFNB28

22q13

TRIOBP

Walsh et al., 2000 ; Shahin et al, 2006 ; Riazuddin et al, 2006

DFNB29

21q22

CLDN14

Wilcox et al., 2001

DFNB30

10p11.1

MYO3A

Walsh et al., 2002

DFNB31

9q32-q34

WHRN

Mustapha et al., 2002 ; Mburu et al., 2003

DFNB32

1p13.3-22.1

GPSM2

Masmoudi et al., 2003 ; Walsh et al., 2010

DFNB33

9q34.3

unknown

Medlej-Hashim et al., 2002

DFNB35

14q24.1-24.3

ESRRB

Ansar et al., 2003 ; Collin et al., 2008

DFNB36

1p36.3

ESPN

Naz et al., 2004

DFNB37

6q13

MYO6

Ahmed et al., 2003

DFNB38

6q26-q27

unknown

Ansar et al., 2003

DFNB39

7q21.1

HGF

Schultz et al., 2009

DFNB40

22q

unknown

Delmaghani et al., 2003

DFNB42

3q13.31-q22.3

ILDR1 

Aslam et al., 2005

Borck et al., 2011

DFNB44

7p14.1-q11.22

unknown

Ansar et al., 2004

DFNB45

1q43-q44

unknown

Bhatti et al., 2008

DFNB46

18p11.32-p11.31

unknown

Mir et al., 2005

DFNB47

2p25.1-p24.3

unknown

Hassan et al., 2005

DFNB48

15q23-q25.1

unknown

Ahmad et al., 2005

DFNB49

5q12.3-q14.1.

MARVELD2

Ramzan et al., 2004 ; Riazuddin et al., 2006

DFNB51

11p13-p12

unknown

Shaikh et al., 2005

DFNB53

6p21.3

COL11A2

Chen et al., 2005

DFNB55

4q12-q13.2

unknown

Irshad et al., 2005

DFNB59

2q31.1-q31.3

PJVK

Delmaghani et al., 2006

DFNB61

7q22.1

SLC26A5

Liu et al., 2003

DFNB62

12p13.2-p11.23

unknown

Ali et al., 2006

DFNB63

11q13.2-q13.4

LRTOMT/COMT2

Du et al., 2008 ; Ahmed et al., 2008 

DFNB65

20q13.2-q13.32

unknown

Tariq et al., 2006

DFNB66

6p21.2-22.3

LHFPL5

Tlili et al., 2005 ; Shabbir et al., 2006 ; Kalay et al., 2006

DFNB67

See DFNB66

 

 

DFNB68

19p13.2

unknown

Santos et al., 2006

DFNB71

8p22-21.3

unknown

Chishti et al., 2009

DFNB72

19p13.3

GIPC3  

Ain et al., 2007 ; Rehman et al., 2011

DFNB73

1p32.3

BSND 

Riazuddin et al., 2009 

DFNB74

12q14.2-q15

MSRB3  

Waryah et al., 2009 ; Ahmed et al., 2011

DFNB77

18q12-q21

LOXHD1 

Grillet et al., 2009

DFNB79

9q34.3

TPRN

Rehman et al., 2010

DFNB81

19p

unknown

Rehman et al., 2011

DFNB82

see DFNB32

 

DFNB83

see DFNA47

 

 

DFNB84

12q21.2

PTPRQ

Schraders er al., 2010

DFNB85

17p12-q11.2

unknown

Shahin et al., 2010

DFNB91

6p25

SERPINB6

Sirmaci et al., 2010

DFNB93

11q12.3-11q13.2

unknown

Tabatabaiefar et al., 2011

DFNB95

19p13

GIPC3  

Charizopoulou et al., 2011

(Adapted from Hereditary Hearing Loss Homepage: http://dnalab-www.uia.ac.be/dnalab/hhh/)

C) X-LINKED NONSYNDROMIC HEARING IMPAIRMENT:

DFN3 (mapped to Xq21.1) is characterized by a mixed conductive- sensorineural hearing loss, the conductive component of which is caused by stapedial fixation. In contrast with other types of conductive hearing loss, surgical correction is not accepted because an abnormal communication between the cerebrospinal fluid and perilymph results in leakage known as "perilymphatic gusher." Thus, a complete loss of hearing occurs when the oval window is fenestrated or removed. Radiology with computed tomography is helpful and demonstrates a dilation of the internal auditory meatus with an abnormal communication between the subarachnoid space and the cochlear endolymph. The causative gene for DFN3 is POU3F4 (Vore et al, 2005). Additional X-linked nonsyndromic hearing loss phenotypes include profound prelingual hearing loss characteristic of both DFN2 and DFN4. DFN6 is characterized by a bilateral high frequency impairment beginning at 5 to 7 years of age and progressing, by adulthood, to severe-to-profound hearing impairment, covering all frequencies.

D) MITOCHONDRIAL NONSYNDROMIC HEARING IMPAIRMENT:

Some mitochondrial DNA mutations cause nonsyndromic hearing loss. In two families, a homoplasmic mutation at nt1555 (A-to-G) in the mitochondrial MTRNR1 gene has been reported. This mutation is also present in persons with aminoglycoside-induced ototoxic hearing loss. Two other families with maternally inherited nonsyndromic hearing loss have been identified with heteroplasmy for an A-to-G transition at nt7445 of the MTTS1 gene. The penetrance of the hearing impairment caused by these mitochondrial mutations is low, suggesting that unidentified genetic or environmental factors play an important role in the progression of the hearing impairment (Fischel, 1998).

2.7 DIAGNOSIS OF DEAFNESS:

Hearing is measured in decibels (dB). The threshold or 0 dB mark for each frequency refers to the level at which normal young adults perceive a tone burst 50% of the time. Hearing is considered normal if an individual's thresholds are within 20 dB of normal thresholds.

2.7.1 PHYSIOLOGIC TESTS:

Physiologic tests objectively determine the functional status of the auditory system and can be performed at any age.

Physiologic tests include:

Auditory brainstem response testing (ABR, also known as BAER, BSER). ABR uses a stimulus (clicks) to evoke electrophysiologic responses, which originate in the VIIIth cranial nerve and auditory brainstem and are recorded with surface electrodes. ABR "wave detection threshold" correlates best with hearing sensitivity in the 1500 to 4000 Hz region in neurologically normal individuals; ABR does not assess low frequency (<1500 Hz) sensitivity.

Evoked otoacoustic emissions (EOAEs). EOAEs are sounds originating within the cochlea that are measured in the external auditory canal using a probe with a microphone and transducer. EOAEs reflect primarily the activity of the outer hair cells of the cochlea across a broad frequency range and are present in ears with hearing sensitivity better than 40-50 dB HL (HL = hearing level).

Immittance testing (tympanometry, acoustic reflex thresholds, acoustic reflex decay). Immittance audiometry assesses the peripheral auditory system, including middle ear pressure, tympanic membrane mobility, eustachian tube function, and mobility of the middle ear ossicles.

2.7.2 AUDIOMETRY

Audiometry subjectively determines how the individual processes auditory information, i.e., hears

Audiometry consists of behavioral testing and pure tone audiometry.

Behavioral testing includes behavioral observation audiometry (BOA) and visual reinforcement audiometry (VRA). BOA is used in infants from birth to six months of age, is highly dependent on the skill of the tester, and is subject to error. VRA is used in children from six months to 2 1/2 years and can provide a reliable, complete audiogram, but is dependent on the child's maturational age and the skill of the tester.

Pure-tone audiometry (air and bone conduction) involves determination of the lowest intensity at which an individual "hears" a pure tone, as a function of frequency (or pitch). Octave frequencies from 250 (close to middle C) to 8000 Hz are tested using earphones. Intensity or loudness is measured in decibels (dB), defined as the ratio between two sound pressures. 0 dB HL is the average threshold for a normal hearing adult; 120 dB HL is so loud as to cause pain. Speech reception thresholds (SRTs) and speech discrimination are assessed.

In air conduction audiometry, sounds are presented through earphones; thresholds depend on the condition of the external ear canal, middle ear, and inner ear.

In bone conduction audiometry, sounds are presented through a vibrator placed on the mastoid bone or forehead, thus bypassing the external and middle ears; thresholds depend on the condition of the inner ear.

Conditioned play audiometry (CPA) is used to test children from ages 2 1/2 to five years. A complete frequency-specific audiogram for each ear can be obtained from a cooperative child.

Conventional audiometry is used to test individuals age five years and older; the individual indicates when the sound is heard.

2.7.3 AUDIOGRAM:

Graphic representation of hearing is called as audiogram. The values are drawn on a graph paper showing the frequencies in Hz on horizontal axis and intensity in dB on vertical axis (Fig. XVII).The symbol used for right ear on audiogram is "O" and for left ear is "X".

The softest sound we are able to hear at each pitch is recorded on the audiogram. The softest sound we are able to hear is called our threshold. Thresholds of 0-25 dB are considered normal (for adults). The audiogram in the Figure: XVI demonstrates the different degrees of hearing loss.

b

Figure: XVI The Audiogram Demonstrates Different Degrees of Hearing Loss

sample audiogram

Figure: XVII The Audiogram Showing Hearing Loss For Left And Right Ear

severe hearing loss audiogram

Figure: XIII The Audiogram Showing Severe Hearing Loss

2.8 TREATMENT OF DEAFNESS:

A) HEARING AIDS:

Patients with hearing problems are recommended to use hearing aids, which are electronic sound-amplifying devices worn in or behind the ear.Hearing aids have the same basic components as any public-address system, but all the components are miniature. A small tube directs the amplified sound from the receiver into the ear canal of the wearer. A unit of this sort can fit into the ear canal with only a small protruding part. If hearing loss is caused by the malformation of the ear canal or impaired function of the middle ear, a small vibrator may b clamped against the mastoid bone behind the ear with a headband; the sound is conducted by the vibrator through the bones of the head to the inner ear.

B) COCHLEAR IMPLANT:

Damage to the organ of Corti in the inner ear accounts either total deafness or severe hearing impairment. Scientists have recently developed an electronic hearing aid called cochlear implant for such profoundly deaf persons whose auditory nerves are functional. This device is more sophisticated than a hearing aid, which merely increases the volume of the sounds that pass through the normal hearing organs.

The cochlear implant works by translating sound waves into electrical signals. These signals are relayed to electrodes that have been surgically implanted in the cochlea so that the auditory nerve is directly stimulated. After successful surgery, once deaf or severely hearing- impaired patients can usually detect a wide range of sounds, but results depend on the factors that include health of the auditory nerves and the duration of deafness.

The device consists of electrodes that are embedded in the cochlea of the inner ear to stimulate the auditory nerve and are connected through the mastoid bone to a receiver surgically placed beneath the skin. A microphone near the ear relays sound signals to a microprocessor, which converts then into electric signals that are sent to a transmitter behind the ear on to the receiver and cochlear electrodes (Fig. XIX).

A microphone picks up the sound waves and transmits them to the speech processor, a miniature computer that turns sounds into digitized electrical signals. These signals travel to a receiver placed beneath the skin and from there to electrodes that have been surgically implanted in the cochlea. In cochlea, the signals stimulate the auditory nerve, which carries the signals to the brain. The brain interprets the electrical signals as sounds.

Figure: XIX Design and Function of the Cochlear Implant

2.9 GENE MAPPING:

Genetic engineering techniques have enabled scientists to determine the chromosomal location and DNA structure of all the genes found within a variety of organisms with the help of gene-mapping techniques, which has two main categories: Linkage or genetic mapping, a method that identifies only the relative order of genes along a chromosome; and physical mapping, more precise method that can place genes at specific distances from one another on a chromosome (Strachan, 2001). Both types of mapping use markers in the DNA sequence, detectable physical or molecular characteristics that differ among individuals and that are passed from one generation to the next.

2.9.1 GENE LINKAGE:

Recent developments in genetic research have accelerated the discovery of individual genes and enhanced our understanding of how genes work and how gene abnormalities lead to disease. There is a growing list of molecular diagnostic tests, with estimates that 10 to 12 new tests become available each year. One of these diagnostic tests is indirect analysis. Indirect analysis often referred to as linkage analysis.

Mendel Gregor Johann (1822-1884), Austrian monk, (whose experimental work became the basis of modern hereditary theory) proposed that two phenotypes occur randomly with respect to one another in a manner known as independent assortment. Today scientists understand that independent assortment occurs when the genes affecting the phenotypes are found on different chromosomes. An exception to independent assortment develops when genes appear near one another on the same chromosomes (Bateson et al., 1905; Dnyansagar, 1989). When genes occur on the same chromosome, they are inherited as a single unit.

Linkage analysis is the technique which is used to determine the genetic location of a disease gene when there is no other indication (no cytogenetic abnormality, co-inherited disorders, good candidate genes, or known protein product) to determine the location of that gene. The goal of linkage analysis is to identify a piece of DNA of known location that is inherited by all family members affected by the disorder being studied, and is not inherited by any of the unaffected family members. This piece of DNA is said to be 'co-inherited' or to 'co-segregate' with the disease phenotype. Once this piece of DNA is found, one knows that the disease gene must lie somewhere close or with in that piece of DNA. Determining the location of the disease gene is the first step toward identifying the gene itself.

The pieces of DNA of known location that are used in linkage analysis are called 'polymorphisms'. Polymorphisms are benign differences in DNA found among humans that are not related to any clinical conditions (they are not disease mutations). Polymorphisms are abundant in the human genome, and can be found between and close to virtually all genes. By identifying a co-inherited polymorphism, testing additional polymorphisms in the vicinity, and then using statistical programs to calculate the odds that you are correct in the choice of location (the LOD score), the disease gene can be placed within a 'critical region' flanked by key polymorphic markers and measured using a genetic distance referred to as a cM (centiMorgan).

Linkage analysis is a relationship between the loci; i.e. two loci on the same chromosome are said to be linked if the phenomenon of crossing over does not separate them. Actually, at the stage of meiosis homologous chromosomes exchange segments as a foundation for the process of recombination or crossing over. If two loci are physically close to each other on the same chromosome then there are rare chances that they will be separated by recombination event. As for this, a crossover will have to occur in small distance between the two loci, which is very rare; the two loci will tend to be inherited together. Sets of alleles for different markers or genes on the same chromosome are termed as haplotypes. Alleles on the same haplotype are passed on in pedigrees as a block. These blocks are only be broken by crossing over. The term linkage refers to the loci, not to specific alleles at these loci. The most common application of linkage analysis is to try and find the location, in the genome, for a gene responsible for a certain mendelianly inherited disease (Ott et.al., 1991).

Alleles at loci on same chromosome for different genes co-segregate at a rate that is associated to the physical distance between them on the chromosome. This rate is the probability or recombination fraction (θ), of a recombination event occurring between two loci. Two loci are said to be genetically linked when recombination fraction is less than 0.5. One of these loci is the disease locus while the other is a polymorphic marker like micro satellite repeats (Strachan and Read et.al, 1996). The object of linkage analysis is to estimate recombination fraction and to test if θ is less than 0.5 between two loci i.e. weather an observed deviation from 50% recombination is statistically significant or not. The recombination fraction ranges from θ=0 for loci right next to each other through θ=0.5 for loci apart (or on different chromosomes), so that it can be taken as a measure of the genetic distance or map distance between gene loci. This measure works well for small distances. The unit of measurement is 1 map unit=1 centimorgan (cM), correspondingly approximately to a recombination fraction of 1%. However, because of the occurrence of multiple crossovers, the recombination fraction is not an additive distance measure and must therefore be transformed by a map function into map distance (Ott et al., 1991).

Recombinants in the pedigrees have to be analyzed to observe the presence or absence of linkage between two loci. It is not usually possible to count these for human pedigrees. For this reason likelihood, methods are used (Strachan and Read et.al, 1996) which calculate the likelihood of a given pedigree under different assumptions about the recombination fraction between two loci. In these calculations, recombination and non-recombination for each possible genotype are calculated. Computers are utilized for this, as the involved calculations are quite complex. A logarithm ratio is calculated (LOD-likelihood of Odds) denoted by Z. LOD score is logarithmic of odds that the loci are linked with recombination fraction θ rather than unlinked (θ = 0.5). A score of +3 or a positive score is an indication of linkage while a score of –2 or a negative score denotes absence of linkage.

It is necessary to have polymorphic markers, which can be checked for inheritance with the disease locus in question for linkage analysis. Micro satellite repeats, particularly dinucleotide and tetra nucleotide repeats are very important in this aspect, as they are highly polymorphic and abundant in the genome. CA/TG repeats are most common accounting for 0.5% of the genome (Strachan and Read et al., 1996).Family size and structure, the number of family members who agree to participate in the linkage study, and accuracy of clinical data from each participant all play a major role in the success of linkage analysis. To obtain a significant LOD score, it is necessary to have a minimal number of family members participating in the study. In addition, for the analysis to be successful, it is also necessary to have accurate clinical information about each participant.

2.9.2 PHYSICAL MAPPING:

It determines the physical distance between landmarks on the chromosomes. The most precise physical mapping techniques combine robotics, laser, and computers to measure the distance between genetic markers. For these maps, DNA is extracted from human chromosomes and randomly broken into many pieces. The DNA fragments are then duplicated numerous times in the laboratory so that the resulting identical copies, called clones, can be tested individually for the presence or absence of specific genetic landmarks. Those clones that share several landmarks are likely to come from overlapping segments of the chromosome. The overlapping regions of the clones can then be compared to determine the overall order of the landmarks along the chromosome and the exact sequence in which the cloned pieces of DNA originally existed in the chromosome

2.10 GENE DISCOVERY IN THE AUDITORY SYSTEM: THE PATH TO IMPROVED DIAGNOSIS AND CLINICAL CARE

Inspection of the genes identified in hearing disorders reveals a great diversity of transcripts, perhaps not surprising due to the large variety of cells and complexity of the inner ear. Certainly, the great degree of genetic heterogeneity reflected in the many different syndromes involving hearing loss and mapped loci is indicative of a large number of genes orchestrating the hearing process. Grouping the genes discovered to be etiologic in deafness disorders into functional categories begins the process of understanding their role in hearing. Knowledge of the pathways in which many of these genes function will be an exciting journey in hearing science; no doubt pathways exist that are not yet imagined. Another important aspect of gene discovery for deafness disorders is that it makes possible the development of diagnostic tests and accurate genetic counseling.

2.10.1 GAP JUNCTION PROTEINS: THE CONNEXINS:

Prominent among the group of genes are those encoding gap junctions. A somewhat surprising finding in the field has been the prevalence of mutations in a single gene encoding the gapjunction protein connexin 26, GJB2, accounting for up to 50% of all cases of autosomal recessive prelingual deafness in tested populations (Estivill et al., 1998 and Loffler et al., 2001). Connexin 26 gap junctions are believed to play a critical role in the recycling of potassium ions from their entry into hair cells during sensory transduction from the endolymph through to the stria vascularis, where other potassium channels pump potassium back into the endolymph. Several recurrent mutations have been found in GJB2 (e.g. 35delG, 167delT, and 235delC).

Besides GJB2, genes for other gap junction proteins have been found to be associated with hearing loss including GJB3 (Xia et al., 1998), GJB6 (Grifa et al., 1999) and GJA1 (Liu et al., 2001).

2.10.2 GENES FOR MAINTENANCE OF HAIR CELL FUNCTION:

Another group of genes of intense interest are those required for survival of sensory hair cells. The POU domain transcription factor gene POU4F3 is required for terminal differentiation and maintenance of inner hair cells and an 8 bp deletion in the POU homeodomain results in progressive hearing loss in DFNA15 (Vahava et al., 1998). Studies of such genes may result in valuable insight into the molecular triggers for hair cell degeneration. Loss of hair cells is presumed to be a fundamental cause of progressive age-related hearing loss (presbycusis) and an understanding of this degenerative process might provide the basis for therapeutic intervention. The recent finding of the transmembrane cochlear-expressed gene TMC1 uncovers a common cause of nonsyndromic recessive deafness in Pakistan and India at the DFNB7/B11 locus on chromosome 9 in bands q13–q21. It is predicted that TMC1 protein may mediate an ion-transport or channel function required for the normal function of hair cells.

2.10.3 MODIFIER GENES:

Molecular analyses of the auditory system have already yielded a number of genes in mice and humans that influence the expression or function of other genes. Studies of these genes are certain to provide insight into the interaction of their gene products. In humans, the locus for a modifier gene (DFNM1) for the deafness haplotype in DFNB26 has been identified on chromosome 1 in band q24 (Riazuddin et al., 2000).

2.10.4 MITOCHONDRIAL GENES:

A variety of mitochondrial disorders have been found to involve hearing loss, perhaps reflecting the highly metabolic state of the hearing process (Fischel-Ghodsian, 1999). Of particular interest has been the A1555 to G mutation in 12S rRNA that is recognized as the most frequent cause of aminoglycoside-induced deafness and as the etiology of a nonsyndromic deafness (Prezant et al., 1993). In a recent study a nearly identical degree of mitochondrial dysfunction was observed in enucleated lymphoblastoid cells derived from both symptomatic and asymptomatic individuals from the same kindred (Guan et al., 2001) supporting the possibility of a nuclear gene in modifying the effect of the mutation.

2.11 DFNB7/11/DFNA36/TMC1:

DFNB7/11 is form of autosomal recessive deafness, mapped on chromosome 9q13-q21. The causative gene is Transmembrane Cochlear Expressed gene 1 (TMC1). Mutations in TMC1 cause autosomal dominant deafness at DFNA36 locus. (Kurima et al, 2002). DFNB7 was mapped in 1995 by studying 2 deafness affected inbreed families. (Jain at el, 1995). Each family showed a signified lod score greater than 4.5

In mice the 'deafness' (dn) locus maps to chromosome 19 and flanking loci are syntenic to human 9q11-q21. The dn mutant shows profound deafness with degeneration of the organ of Corti, stria vascularis, and occasionally the saccular macula, starting at about 10 days after birth. The dn mouse is a possible model for DFNB7. (Vreugde et al., 2002).

Linkage analysis in 2 inbred Bedouin kindreds with autosomal recessive nonsyndromic hearing loss revealed evidence for linkage with the markers D9S922 and D9S301 in chromosome 9q (Scott et al.,1996). Genotyping of individuals confirmed linkage with a maximum combined lod score of 26.2 at theta = 0.025 with the marker D9S927. The disease locus was mapped to the interval 9q13-q21. Although this cytogenetic map location is the same as that of DFNB7, the disease interval defined in the kindreds reported by Scott et al., (1996) lies between D9S15 and D9S927, with D9S15 defining the centromeric border. In the kindred reported by Jain et al., (1995) the DFNB7 interval was located between D9S50 and D9S15, with D9S15 defining the most telomeric border.

(Van Camp et al., 1997) referred to the deafness in the family reported by Scott et al., (1996) as DFNB11. Since the phenotype and chromosomal map location are the same as in the family reported by Jain et al., (1995), they may have the same disorder.

(Kurima et al., 2002) used positional cloning to identify the TMC1 gene as the site of mutations causing autosomal recessive deafness that map to 9q13-q21.

2.11.1: POSITIONAL CLONING OF TMC1

Kurima et al., (2002) identified the mutant TMC1 gene in the form of autosomal dominant deafness (DFNA36) and of recessive deafness (DFNB7/11) that map to the same interval on chromosome 9 by positional cloning. The author sequenced several candidate genes and found no mutation. They initiated systematic BLAST analysis of segments of genomic DNA sequence in the critical region to identify additional DFNA36/DFNB7/B11 candidate gene. One sequence was found to be similar to a predicted gene TMC2 on 20p13. They used conserved sequence between TMC2 and the query sequence on chromosome 9q13-q21 to design primers for amplifying potential TMC1 transcr