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聽力障礙是最常見的出生缺陷之一，大約每一千個新生兒中就有三個表現為聽力障礙。世界衛生組織的一份最新報告顯示，世界範圍內有三億六千萬人有大於或等於30 分貝聽閾值的聽損。在中國，根據第二次國家殘疾調查數據估計有兩千萬人群已經診斷為聽力障礙。約佔中國總人口的百分之一點五。其中有十一萬伍仟小於七歲的兒童診斷為重度失聰， 且每年至少有三萬名新生兒有聽力障礙。 / 遺傳性耳聾可根據臨床表現診斷。然而導致耳聾的遺傳背景卻千差萬別。遺傳因素約佔學語前性耳聾的百分之五十。明確導致耳聾的遺傳性致病因素有助於解釋疾病的發生發展，臨床診療決策，針對病人及家屬有效的遺傳咨詢。然而在臨床上開展針對遺傳性耳聾的分子診斷確非易事。 / 根據文獻報導不同種族耳聾患者尤其獨特的熱點突變類型。了解中國人群的突變種類和人群攜帶率有助於開展耳聾的分子診斷。經過針對中國耳聾患者文獻報導的查詢，我們發現15 個常見突變。並在中國新生兒人群中採用SNaPshot 方法鑑定這15 個突變的基因型。由於Sanger 測序方法所能檢測該方法所能檢測的基因型有限，考慮到遺傳性耳聾的致病基因眾多，我們將液相捕獲和二代測序相結合來實現高通量篩查。針對二百多個耳聾相關基因設計捕獲探針，雜交捕穫後同時進行二代測序。針對該流程自設的生物信息分析方法可有效定位變異位點，且sanger 測序驗證率高。我們亦同時設計了NimbleGen 和Agilent 兩種平台的液相捕獲探針文庫。最終在七名患者中的兩名找到可能與耳聾相關的遺傳突變。 / 伴隨著分子診斷對耳聾患者的廣泛應用，產前診斷需求亦有增加。傳統產前診斷需要有創操作獲取胎兒組織。這些操作可能對胎兒不利從而導致流產。我們將母親血漿DNA 分析胎兒基因型的無創方法应用在一例遺傳性耳聾的產前診斷。胎兒經過分析為致病突變攜帶者。該結果與孕中期有創產前基因檢查結果一致。 / 總而言之，該論文中所總結的耳聾熱點突變的中國人群攜帶率有助於耳聾患者的風險評估和遺傳咨詢。目標區域富集結合二代測序以及無創單倍體分型的方法建立都將增強遺傳性耳聾的基因診斷的應用。 / Hearing loss is one of the most common birth defects, with a prevalence rate of approximately 3 per 1000 neonates. In the latest WHO report, 360 million people worldwide have hearing loss at least greater than 30dB. In China, based on the data of the Second China National Sample Survey on Disability, it was estimated that 20 million people were diagnosed with hearing disability. This accounts for 1.5% of the total Chinese population, and represents 115 000 children under the age of 7 years with severe-to-profound deafness and 30 000 newborns (~0.15%) each year with hearing impairment. / Hereditary hearing loss is due to genetic defects. Therefore establishing a genetic diagnosis for hereditary hearing loss is favorable for explanation of the disease, determination of clinical management, and to offer appropriate genetic counseling for patients and their family members. It has a well-recognized phenotype which is relatively easy to be diagnosed clinically. Nonetheless, genetic causes contributing to hearing impairment are unparalleled heterogeneous. Implementing genetic diagnosis in clinical practice for hereditary hearing loss is challenging. / It has been reported that different ethnic groups show a unique spectrum of common mutations of hearing loss genes. Understanding the spectrum and carrier frequency of common mutations particularly in the Chinese population could facilitate the molecular diagnosis of hearing loss. According to common mutations extracted from the literatures regarding hearing loss patients, we studied the carrier frequency of 15 common mutations in GJB2, SLC26A4 and the mitochondrial genome by the SNapShot method. This is the first epidemiological study of nonsyndromic hearing loss in Chinese that reveals high carrier frequencies (one per 6.3 newborns) of 15 common mutations in newborns. / Considering the limitation of mutations screened by Sanger sequencing, we developed the solution-based capture system with massively parallel sequencing (MPS) to target more than 252 genes related with hearing loss. An in-house bioinfomatic analysis pipeline was constructed to identify candidate variants with a high validation rate. We designed both Agilent SureSelect and NimbleGen SeqCap target enrichment platforms and adopted them for the mutation discovery of hearing loss genes in unrelated Chinese families with nonsyndromic hearing loss. Our targeted genome enrichment (TGE) MPS approach identified an additional 28% (2 out of 7) of unrelated Chinese families carrying novel mutations. / As more hearing loss patients receive a genetic diagnosis, there will be a foreseeable growing demand for prenatal diagnosis. Standard prenatal testing requires an invasive procedure to obtain a fetal sample, which has the risk of fetal loss. Therefore we explored the application of a noninvasive method using cell free fetal DNA in a family with a GJB2 mutation. Using haplotype analysis of a 1.1 Mb flanking region around GJB2, we successfully recovered the fetal haplotype and deduced that the fetus inherited the paternal mutant allele and maternal wide-type allele. / In summary, our carrier frequency data aid in effective risk assessment and genetic counseling for hearing loss patients in the Chinese population. The newly established target genome enrichment MPS method and noninvasive haplotype approach enhanced the success of genetic diagnosis of hereditary hearing loss. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Cao, Ye. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 116-130). / Abstracts also in Chinese.

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The most common inherited sensory disorder that affects I in 1 000 children is severe hearing loss. In developed countries, about a third of cases have a genetic origin, 80% of which are autosomal recessive forms (DFNB). Before 1993 few genes causing hearing loss had been identified, but since then a large number of genes related to this problem have been identified. Studies indicate that the DFNBI locus, located at position 13q11-12, contributes to 20% of all childhood deafness and may have a carrier rate as high as 2.8%. There are two genes linked to DFNB 1, GJB2 and GJB6, which are the major genetic cause of non-syndromic autosomal recessive deafness. GJB2 and GJB6 encode the connexin proteins connexin 26 and 30 (Cx26 and Cx30), respectively. The specific aim of this study was to determine the role of GJB2 and GJB6 in deafness within the South African population, since there are no published results involving South African patients with non-syndromic autosomal recessive deafness. This study therefore involved the identification of mutations within the coding region of the GJB2 and GJB6 genes in the South African population and the determination of their specific allele frequencies. Another aim of this study was to analyse the effectiveness of three single-strand conformation polymorphic (SSCP) gel electrophoresis systems in the detection of GJB2 mutations, for use in a standardised diagnostic program. A total of 44 families were recruited and divided into either the familial or sporadic study group, which consisted of 16 and 28 families, respectively. Control samples were also screened from 50 Caucasians and 50 Mixed Ancestry individuals collected from the general population. To achieve the aims of this study, polymerase chain reaction (PCR) amplification followed by automated DNA sequencing of the coding regions of GJB2 and GJB6 was performed. The three SSCP systems that were tested for their effectiveness in detecting mutations within the coding region of GJB2 included mini polyacrylamide, SSCP-urea and two buffer gel electrophoresis systems. In total, six previously reported mutations (35delG, 312de1l4, W24X, M34T, V37I and W44X), a novel mutation (N62I), and four benign polymorphisms (V27I, A40A, R127H and V153I) were detected in GJB2. In the GJB6 gene only the S199T polymorphism was observed. It was determined that the most common mutations found within the Caucasian and Mixed Ancestry populations of South Africa were 35delG and 312de1l4 of GJB2. An overall detection rate of 35.227% was achieved in non-syndromic autosomal recessive deafness amongst this patient cohort. It was also observed that none of the SSCP gel electrophoresis systems were effective at detecting all of the GJB2 mutations. This could change if the systems were specifically optimised for the cornmon mutations that were identified. This study therefore, provides information that can be used in the formulation of a screenmg program for non-syndromic autosomal recessive deafness specific to the South African population. Further research should be conducted involving other genes, in addition other population groups of South Africa to provide a more comprehensive genetic diagnostic and counselling tool. / AFRIKAANSE OPSOMMING: Die mees algemene oorerflike sensoriese steuring wat 1 in 1 000 kinders affekteer is ernstige gehoorverlies. In ontwikkelde lande het omtrent een-derde van die gevalle 'n genetiese oorsprong, waarvan 80% outosomaal resessiewe vorms is (DFNB). Tot en met 1993 is min gene wat gehoorverlies veroorsaak geïdentifiseer, maar sedertdien is 'n groot aantal gene gelokaliseer en verskeie is ook al gekloneer. Studies toon dat die DFNB 1 loci, wat in posisie 13q 11-12 gevind word, 20% van doofheid in kinders veroorsaak, en dit het 'n draer frekwensie van so hoog as 2.8%. Twee gene wat koppeling met DFNBI toon, GJB2 en GJB6, is die vernaamste genetiese oorsaak van nie-sindromise autosomaal resessiewe doofheid. GJB2 en GJB6 koder vir die connexin proteïne 26 en 30 (Cx26 en Cx30), onderskeidelik. Die spesifieke doel van hierdie studie is om die rol van GJR2 en GJB6 in doofheid binne die Suid- Afrikaanse populasie te bepaal, aangesien daar tans nog geen gepubliseerde resultate omtrent Suid- Afrikaanse pasiënte met nie-sindromiese outosomaal resessiewe doofheid is nie. Hierdie studie handel dus oor die identifikasie van mutasies wat binne die koderende areas van die GJR2 en GJB6 gene voorkom in die Suid-Afrikaanse populasie, asook oor die bepaling van hulle spesifieke alleel frekwensies. Verder het hierdie studie ten doelom die effektiwiteit van drie enkel-string konformasie polimorfisme (SSCP) gel-elektroforese metodes in die opsporing van GJB2 mutasies te analiseer met die oog op toekomstige gebruik in 'n gestandardiseerde diagnostiese program. Altesaam 44 families is ingesamel en gekategoriseer in familiële of sporadiese studie-groepe met 16 en 28 families onderskeidelik. Kontrole monsters van 50 Kaukasiese en 50 Gemengde Herkoms individule uit die algemene populasie is ook getoets. Om die doeleindes van die studie te bereik is PKR amplifikasie en outomatiese DNS volgordebepaling van die koderende area van GJB2 en GJR6 gedoen. Die drie SSCP sisteme wat getoets is vir hulle effektiwiteit in die identifisering van mutasies in die koderende area van GJB2 sluit in mini poli-akrielamied, urea en twee-buffer gel elektroforese sisteme. In totaal is ses gerapporteerde mutasies (35delG, 312de114, W24X, M34T, V37I en W44X), 'n nuwe mutasie (N62I), en vier onskadelike polimorfismes (V27I, A40A, R127H en V153I) opgespoor in GJB2, maar in GJB6 is net die S199T polimorfisme waargeneem. Uit die resultate kon afgelei word dat 35deiG en 312de114 van GJB2 die mees algemene mutasies binne die Kaukasiese en Gemengde Herkoms bevolkings van Suid Afrika is. Die total ontdekking standaard van 35.227%· vir nie-sindromise autosomaal resessiewe doofheid tussen herdie patient kohort was bereik. Verder is waargeneem dat geen van die SSCP gel elektroforese metodes effektief was om al die mutasies van GJB2 op te spoor nie. Die situasie kan egter verander as die sisteme spesifiek geoptimiseer word vir die algemene mutasies wat gevind is. Hierdie studie verskaf dus inligting wat gebruik kan word in die verskaffing van 'n diagnostise program vir nie-sindromise outosomaal resessiewe doofheid wat spesifiek is vir die Suid- Afrikaanse populasie. Verdere navorsing wat ander gene en ander populasie groepe van Suid-Afrika insluit, behoort egter uitgevoer te word om uiteindelik 'n meer uitgebreide genetiese diagnostiese en raadgewing diens daar te stel.

Iroquois genes encode a family of highly conserved TALE homeodomain transcription factors that are involved in multiple developmental processes. Physiological tests indicated that Irx3 and Irx5 mutant mice displayed hearing impairment. However, the functions of these two genes during inner ear development are not known. The aim of this study is to characterize the roles of Irx3 and Irx5 during mammalian inner ear development using mouse models, in order to reveal the underlying mechanism for the hearing abnormality in the mutants. Two mouse mutants, Irx3tauLacZ and Irx3flox5EGFP with β-gal and EGFP reporters, were analyzed to examine the expression of these two genes in the otic vesicle and cochlear epithelium. In the otocyst, both Irx3 and Irx5 were expressed in the ventral-medial region. Irx5 expression was restricted to the non-sensory domain of the cochlear epithelia, while Irx3 was widely expressed, including the auditory sensory organ, the organ of Corti. The overlapping expression patterns of Irx3 and Irx5 suggest that they may share redundant functions. To investigate the roles of Irx3 and Irx5 during inner ear development, phenotypic analysis was performed on Irx3-/-, Irx5-/- and Irx3/5-/- mutant embryos. As shown by paint-filling analysis, Irx3/5-/- displayed shortened cochlear duct, enlarged cochlear lumen with fused sensory organ. Whole-mount phalloidin staining of hair cell bundles showed that Irx3-/- displayed occasional ectopic inner hair cells. Moreover, only supernumerary vestibular hair cell-like cells were developed in Irx3/5-/- mutant. These results suggest that Irx3 and Irx5 are required for inner ear morphogenesis and the formation of organ of Corti. To understand the effect of Irx3 and Irx5 in the cellular patterning of the cochlea, mutant cochleae were analyzed with markers for different regions of the cochlear epithelia. Altered expression domain of MyoVIIa, Sox2 and Gata2 in Irx3/5-/- cochlea revealed that the boundary between the Kolliker’s organ and the organ of Corti was lost and the location of sensory and non-sensory region was shifted. These results imply that Irx3 and Irx5 function in the establishment of the sensory/non-sensory boundary. It is known that p27kip1 regulates the wave of cell cycle exit in the developing organ of Corti and Sox2 takes part in prosensory specification. To explore the underlying reason for the patterning defects in Irx3/5-/- mutant, cochlear duct from prosensory stages were analyzed. Irx3/5-/- showed altered Sox2 and p27kip1 expression, with expanded prosensory domain and disrupted cell cycle exit. Ectopic prosensory proliferation was detected in the middle turn of the cochlear duct at E13.5 by BrdU incorporation assay. Therefore, Irx3 and Irx5 may participate in the subdivision of sensory territory in developing cochlea by controlling prosensory proliferation. In summary, this study demonstrates that Irx3 and Irx5 cooperate in multiple aspects of inner ear development: an early role to regulate prosensory proliferation and cell cycle exit; a second role to regulate cellular patterning of the cochlear duct by controlling the setting of sensory/non-sensory boundaries in the cochlea; a later role to regulate inner ear morphogenesis. This study supports the idea that Irx3 and Irx5 act as patterning genes during vertebrate evolution. / published_or_final_version / Biochemistry / Master / Master of Philosophy

Sox10 is a high mobility group (HMG) domain transcription factor which is an important regulator for neural crest development. SOX10 mutations have been identified in Waardenburg-Shah syndrome type 4 (WS4) patients who suffer from sensorineural deafness. However, the mechanisms underlying the hearing defect of SOX10-mediated WS4 are unclear. The aim of this study is to elucidate the function of Sox10 during mouse inner ear development using a mutant mouse model, in order to reveal the underlying basis for SOX10 mutation associated sensorineural deafness in WS4 patients. The mammalian inner ear originates from the otic placode epithelium as well as neural crest cells (NCCs). To understand the role of Sox10 in inner development, I investigated the contribution of cranial NCCs to the cochleovestibular ganglion (CVG) by lineage tracing analysis, using Wnt1-cre;ZEG mice in which all NCCs were marked by GFP. Co-expression of GFP-positive cells with the glial marker BFABP suggested that glial cells in the CVG were derived from NCCs. Furthermore, Sox10-expressing NCCs were found to invade the CVG at 30-somite stage. These results suggest a role of Sox10 in regulating cranial NCCs contribution to CVG glia. In our laboratory we have generated a mouse mutant Sox10EGFP in which the Sox10 N-terminal domain was fused to the EGFP reporter. To investigate the function of Sox10 in NCCs invasion and gliogenesis of CVG, phenotypic analysis of Sox10NGFP mutant mouse were performed. EGFP expression in the CVG and inner ear epithelium of Sox10NGFP/+ embryos recapitulated the dynamic expression pattern of Sox10. Sox10NGFP/NGFP mutants displayed a reduced number of migrating NCCs and lacked NCCs or glia in their CVG. Moreover, loss of glial cell in the developing spiral ganglia of Sox10NGFP/NGFP mice led to disorganized fasciculation and degeneration of axonal filaments. These data suggest that Sox10 is required for maintaining the cranial NC stem cell pool, and is also essential for CVG gliogenesis and normal growth and innervation of spiral ganglion neurons. To study the function of Sox10 in regulating cochlear morphogenesis, morphological and histological analysis of mutant cochlear were performed. As illustrated by paint-filling analysis, Sox10NGFP/NGFP mice developed a shortened cochlear duct, reduced cochlear turning and enlarged endolymph lumen. Sensory hair cell patterning in the organ of Corti was normal in the Sox10 mutant as shown by immunohistochemistry analysis, suggesting that cochlear lumen enlargement was not due to disrupted planar cell polarity (PCP) pathway. To explore the molecular basis of Sox10-mediated cochlear morphogenic defect, expression of genes related to cochlear development were examined by qRT-PCR. Candidate genes included those involved in fluid homeostasis, which are known to affect the size of cochlear lumen. Up-regulated expression of Aquaporin 3, a water channel protein in the cochlear epithelium that facilitates water transport across the cell membrane, was observed in Sox10NGFP/NGFP cochlear. These results suggest that Sox10 may regulate cochlear morphogenesis by controlling endolymph homeostasis. In conclusion, Sox10 is required in multiple processes during inner ear development including NCC invasion, gliogenesis and cochlear morphogenesis, and their abnormal development can lead to sensorineural deafness in WS4 syndrome. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

abstract / Biochemistry / Master / Master of Philosophy

The audiological characteristics of the Monge family of Costa Rica were investigated in a sample of fifty-two affected members and twelve unaffected members. Through laboratory analysis by staff personnel from the University of Costa Rica and audiological test results obtained in the present investigation, it was concluded that affected Monge members demonstrate a slowly progressive low frequency sensorineural hearing loss of autosomal dominant transmission. The initial site of lesion appears to be the apical portion of the cochlea, with significant onset occurring during early childhood following normal speech and language acquisition. The rate at which the hearing loss progresses and the frequency regions affected are contingent upon chronological age, culminating in a flat profound hearing impairment by age thirty for all affected members.

La seqüenciació del genoma humà ha marcat una fita important en la història de la biologia. Com a conseqüència, la genètica i la genòmica han experimentat un progrés enorme. Això ha permès un millor coneixement tant de les causes genètiques de malalties humanes, com del per què de les diferències comunes entre individus. Com a sistemes complexos que som tots els éssers vius, hem de considerar el paper que tenen les interaccions entre les diferents parts del genoma a l'hora d'especificar el resultat final, és a dir, el fenotip. Igualment, podem dir que el genoma conté un conjunt d'instruccions, però que la forma en què aquestes es porten a terme depèn, també de contingències ambientals i històriques. Per tant, la naturalesa de les instruccions genètiques no és completament determinista en tots els casos, si bé hi ha una sèrie de processos en què sí que es compleix aquesta perfecta relació entre herència i expressió final. Aquesta mateixa situació es presenta amb certes alteracions genètiques i amb el desenvolupament de patologies, la qual cosa facilita enormement el diagnòstic precoç i obre les possibilitats per a la teràpia genètica. Però la gran majoria de fenotips, incloent-hi moltes condicions d'interès per a la medicina, tenen una base complexa, és a dir, no existeix "el gen" que determina el caràcter de forma unívoca, sinó que aquest és el resultat de l'acció simultània de molts gens, no tots amb la mateixa participació, juntament amb l'efecte de l'ambient. Aquesta tesi doctoral va arrencar en aquest punt, tenint com a objectiu l'aprofundiment en les bases genètiques d'un tipus de sordesa lligada a mutacions al vi Preface DNA mitocondrial i de la qual se'n tenien evidències de la implicació tant de factors ambientals com diversos factors genètics. D'altra banda, els tests basats en l'ADN són un dels primers usos comercials i d'aplicació mèdica d'aquests nous descobriments de la genètica. Aquests tests poden ser utilitzats per al diagnòstic de malalties, confirmació diagnòstica, informació del pronòstic, així com del curs de la malaltia, confirmar la presència de malaltia en pacients assimptomàtics i amb diferents graus de certesa, predir el risc de futures malalties en persones sanes i en la seva descendència. Aquest és l'objectiu final, i sovint encara utòpic, de la recerca en biomedicina: una millor comprensió del procés biològic, que derivi en un millor tractament i prevenció de la malaltia. Aquesta tesi també ha volgut contribuir humilment en aquest aspecte. Durant aquests anys s'han recollit centenars de mostres de famílies sordes, amb la finalitat de donar un "diagnòstic" de la causa genètica. Poques vegades ho hem conseguit, però en qualsevol cas, si això alguna vegada ha ajudat a algú d'alguna manera, ja em dono per satisfeta.