Rachel Fisher

Mutations in the connexin 26 gene (GJB2) among Ashkenazi jews with nonsyndromic recessive deafness

BACKGROUND Mutations in the GJB2 gene cause one form of nonsyndromic recessive deafness. Among Mediterranean Europeans, more than 80 percent of cases of nonsyndromic recessive deafness result from inheritance of the 30delG mutant allele of GJB2. We assessed the contribution of mutations in GJB2 to the prevalence of the condition among Ashkenazi Jews. METHODS We tested for mutations in GJB2 in DNA samples from three Ashkenazi Jewish families with nonsyndromic recessive deafness, from Ashkenazi Jewish persons seeking carrier testing for other conditions, and from members of other ethnic groups. The hearing of persons who were heterozygous for mutations in GJB2 was assessed by means of pure-tone audiometry, measurement of middle-ear immittance, and recording of otoacoustic emissions. RESULTS Two frame-shift mutations in GJB2, 167delT and 30delG, were observed in the families with nonsyndromic recessive deafness. In the Ashkenazi Jewish population the prevalence of heterozygosity for 167delT, which is rare in the general population, was 4.03 percent (95 percent confidence interval, 2.5 to 6.0 percent), and for 30delG the prevalence was 0.73 percent (95 percent confidence interval, 0.2 to 1.8 percent). Genetic-linkage analysis showed conservation of the haplotype for 167delT but the existence of several haplotypes for 30delG. Audiologic examination of carriers of the mutant alleles who had normal hearing revealed subtle differences in their otoacoustic emissions, suggesting that the expression of mutations in GJB2 may be semidominant. CONCLUSIONS The high frequency of carriers of mutations in GJB2 (4.76 percent) predicts a prevalence of 1 deaf person among 1765 people, which may account for the majority of cases of nonsyndromic recessive deafness in the Ashkenazi Jewish population. Conservation of the haplotype flanking the 167delT mutation suggests that this allele has a single origin, whereas the multiple haplotypes with the 30delG mutation suggest that this site is a hot spot for recurrent mutations.

Mutations in the γ-Actin Gene (ACTG1) Are Associated with Dominant Progressive Deafness (DFNA20/26)

Age-related hearing loss (presbycusis) is a significant problem in the population. The genetic contribution to age-related hearing loss is estimated to be 40%–50%. Gene mutations that cause nonsyndromic progressive hearing loss with early onset may provide insight into the etiology of presbycusis. We have identified four families segregating an autosomal dominant, progressive, sensorineural hearing loss phenotype that has been linked to chromosome 17q25.3. The critical interval containing the causative gene was narrowed to ∼2 million bp between markers D17S914 and D17S668. Cochlear-expressed genes were sequenced in affected family members. Sequence analysis of the γ-actin gene (ACTG1) revealed missense mutations in highly conserved actin domains in all four families. These mutations change amino acids that are conserved in all actins, from protozoa to mammals, and were not found in >100 chromosomes from normal hearing individuals. Much of the specialized ultrastructural organization of the cells in the cochlea is based on the actin cytoskeleton. Many of the mutations known to cause either syndromic or nonsyndromic deafness occur in genes that interact with actin (e.g., the myosins, espin, and harmonin). The mutations we have identified are in various binding domains of actin and are predicted to mildly interfere with bundling, gelation, polymerization, or myosin movement and may cause hearing loss by hindering the repair or stability of cochlear cell structures damaged by noise or aging. This is the first description of a mutation in cytoskeletal, or nonmuscle, actin.

Depressive symptoms in mid-pregnancy, lifetime stressors and the 5-HTTLPR genotype

Few studies of gene–environment interactions for the serotonin transporter promoter polymorphism (5‐HTTLPR), life stressors and depression have considered women separately or examined specific types of stressful life events. None have looked at depression during pregnancy. In the Pregnancy Outcomes and Community Health (POUCH) Study, women were queried about history of stressful life events and depressive symptoms at the time of enrollment (15–27 weeks gestation). Stressful life events were grouped a priori into “subconstructs” (e.g. economic, legal, abuse, loss) and evaluated by subconstruct, total subconstruct score and total stressful life event score. The effect of genotype on the association between stressful life events and elevated depressive symptoms was assessed in 568 white non‐Hispanic participants. The relationship between exposure to abuse and elevated depressive symptoms was more pronounced in the s/s group (OR = 24.5) than in the s/l group (OR = 3.0) and the l/l group (OR = 7.7), but this significant interaction was detected only after excluding 73 (13%) women with recent use of psychotropic medications. There was no evidence of gene–environment interaction in analytic models with other stressful life events subconstructs, total subconstruct score or total stressful life events score. These data offer modest support to other reports of gene–environment interaction and highlight the importance of considering specific stressful life events.

Expression of GJB2 and GJB6 Is Reduced in a Novel DFNB1 Allele

In a large kindred of German descent, we found a novel allele that segregates with deafness when present in trans with the 35delG allele of GJB2. Qualitative polymerase chain reaction-based allele-specific expression assays showed that expression of both GJB2 and GJB6 from the novel allele is dramatically reduced. This is the first evidence of a deafness-associated regulatory mutation of GJB2 and of potential coregulation of GJB2 and GJB6.

A NOVEL DFNB1 DELETION ALLELE SUPPORTS THE EXISTENCE OF A DISTANT CIS-REGULATORY REGION THAT CONTROLS GJB2 AND GJB6 EXPRESSION

Wilch E, Azaiez H, Fisher RA, Elfenbein J, Murgia A, Birkenhäger R, Bolz HJ, da Silva‐Costa SM, del Castillo I, Haaf T, Hoefsloot L, Kremer H, Kubisch C, Le Marechal C, Pandya A, Sartorato EL, Schneider E, Van Camp G, Wuyts W, Smith RJH, Friderici KH. A novel DFNB1 deletion allele supports the existence of a distant cis‐regulatory region that controls GJB2 and GJB6 expression.

Interplay of cytokine polymorphisms and bacterial vaginosis in the etiology of preterm delivery.

Recent findings suggest that the association between inflammation-related genes and preterm delivery may be stronger in the presence of bacterial vaginosis (BV). Tumor necrosis factor-alpha (TNFα) and interleukin 1-beta (IL-1β) are pro-inflammatory cytokines capable of inducing preterm labor in non-human primates. In this study the authors tested associations among two TNFα promoter polymorphisms (-G308A and -G238A), a single IL-1β polymorphism (+C3954T), vaginal microbial findings, and risk of preterm delivery. Data were from the Pregnancy Outcomes and Community Health (POUCH) Study (n=777 term and n=230 preterm deliveries). Vaginal smears collected at mid-pregnancy (15-27 weeks gestation) were scored according to Nugents criteria. A Nugent score of ≥ 4 was modeled as the cut-point for intermediate and positive BV. Logistic regression was used to estimate odds ratios for associations among independent covariates (vaginal flora, genotype) and preterm delivery. Results showed that women with a Nugent score of≥ 4 and the TNFα -238 A/G or A/A were at increased risk of delivering preterm (race/ethnicity adjusted OR 2.6, 95% CI 1.2, 5.8). The p-value for the genotype and Nugent score interaction=0.02. This study points to one more example of a potential gene-environment interaction in a preterm delivery pathway. Future tests of this finding will determine the robustness of these results.

A comparison of statistical methods for prenatal screening for Down Syndrome

Currently, prenatal screening for Down Syndrome (DS) uses the mothers age as well as three biochemical markers for risk prediction. Risk calculations for the biochemical markers use a quadratic discriminant function. In this paper we compare several classification procedures to quadratic discrimination methods for biochemical-based DS risk prediction, based on data from a prospective multicentre prenatal screening study. We investigate alternative methods including linear discriminant methods, logistic regression methods, neural network methods, and classification and regression-tree methods. Several experiments are performed, and in each experiment resampling methods are used to create training and testing data sets. The procedures on the test data set are summarized by the area under their receiver operating characteristic curves. In each experiment this process is repeated 500 times and then the classification procedures are compared. We find that several methods are superior to the currently used quadratic discriminant method for risk estimation for these data. The implications of these results for prenatal screening programs are discussed.

Recent Advances in the Understanding of Syndromic Forms of Hearing Loss

There are hundreds of different syndromes that include an auditory phenotype as a prominent feature and nearly as many reviews of this topic (Ahmed, Riazuddin, Riazuddin, & Wilcox, 2003; Griffith & Friedman, 2002; Gurtler & Lalwani, 2002; Lalwani, 2002; Morton, 2002; Petit, 2001; Steel, Erven, & Kiernan, 2002). Approximately 30% of individuals with hereditary hearing loss also have abnormalities of other organ systems and are considered to have a syndromic form of deafness (Gorlin, Toriello, & Cohen, 1995). The accompanying abnormalities range from subtle to obvious and may be congenital or delayed in appearance. The majority of these syndromes are inherited as monogenic disorders. Some of these genes have been mapped to a chromosomal map position and a subset of these mapped genes have been identified (cloned). In Table 1 we list many of the syndromic forms of hearing loss and some of the distinguishing clinical features. In many cases where hearing loss is listed as a part of a syndrome, the loss develops late and may simply be secondary to the general neurological decay. For virtually all inherited syndromic forms of hearing loss the Online Mendelian Inheritance in Man (www.ncbi.nlm.nih.gov/Omim/) has comprehensive descriptions of the clinical features and molecular genetics as well as an all-inclusive list of references. In this review we discuss only advances in our understanding of syndromic forms of deafness that have been made in the past few years. Several deafness syndromes are named after the clinician(s) who first or more fully described the disorder such as Jervell and Lange-Nielsen syndrome, Marshall syndrome, Stickler syndrome, Usher syndrome and Waardenburg syndrome (Table 1). Other hereditary deafness syndromes have been given labels that encompass part or all of the clinical presentation (phenotype) such as Branchio-Oto-Renal syndrome (abbreviated BOR, Table 1). As might be expected, there is a gene for each of these clinically distinct inherited syndromes that include hearing loss as one feature. However, there are exceptions to this generalization. Two phenotypically distinct syndromes may be due to different mutations of the same gene. Geneticists refer to different mutations of the same gene as allelic mutations or, more simply, as alleles. There are many examples of phenotypically distinct syndromes that are caused by allelic mutations such as Marshall syndrome (OMIM 154780) and Stickler syndrome type 2 (OMIM 604841), both of which can be caused by mutations of COL11A1 on chromosome 1p21 (Griffith, Sprunger, Sirko-Osadsa, Tiller, Meisler, & Warman, 1998) (Table 1). Another example is Waardenburg syndrome type 1 (OMIM 193500), Waardenburg syndrome type 3 (OMIM 148820) and Craniofacial-Deafness-Hand syndrome (OMIM 122880), which are clinically distinct but, in fact, are caused by allelic mutations of PAX3 on chromosome 2q35 (Asher, Sommer, Morell, & Friedman, 1996). Moreover, the converse is also true. Mutations of more than one gene may cause the identical clinical phenotype. This is referred to as genetic heterogeneity. For example, Usher syndrome type 1 is characterized by congenital, severe to profound hearing loss, retinitis pigmentosa (RP) with prepubertal onset and vestibular areflexia. Surprisingly, there are at least seven different genes that can cause clinically indistinguishable Usher syndrome type 1 (Table 1). Sometimes a patient has hearing loss that is obvious while their other associated abnormalities escape notice. There are many different reasons for incomplete diagnoses. Two examples illustrate this point. The hearing loss in Usher syndrome types 1 and 2 is congenital, while the onset of RP may be delayed and not noticed until adolescence. Patients who have Jervell and Lange-Nielsen syndrome are hearing impaired, but their heart conduction problems are easily overlooked (Table 1). Therefore, Section on Human Genetics (T.B.F., J.M.S., T.B-Y., A.L., E.R.W., S.R., Z.A., I.A.B.), Section on Gene Structure and Function (S.P.P, A.J.G.), and Hearing Section (S.P.P., A.J.G.), National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Maryland; and Department of Pediatrics and Human Development (R.A.F.), Michigan State University, East Lansing, Michigan.

Polymorphisms in thrombophilia and renin–angiotensin system pathways, preterm delivery, and evidence of placental hemorrhage

OBJECTIVE The purpose of this study was to analyze functional polymorphisms in candidate genes (methylenetetrahydrofolate reductase [MTHFR]677C>T, MTHFR1298A>C, factor 5 1691G>A [FVL], and angiotensinogen (AGT)-6G>A) in relation to a hypothesized placental hemorrhage pathway to preterm delivery (PTD). STUDY DESIGN We assessed maternal genotypes, pregnancy outcomes, and placental pathologic evidence among 560 white and 399 black women who were recruited at mid trimester into a prospective cohort study (1998-2004). Odds of dominant genotypes were calculated for PTDs with (n = 56) or without (n = 177) evidence of placental hemorrhage (referent = term) with the use of race-stratified polytomous logistic regression models. RESULTS Among white women, FVL GA/AA and AGT(-6) GA/AA were both associated with hemorrhage-related PTDs (odds ratio [OR], 4.8; 95% confidence interval [CI], 1.6-14.2 and OR, 3.8; 95% CI, 1.3-10.5, respectively), but not other PTDs (ORs, 1.2 and 0.9, respectively). FVL GA/AA was associated with placental abruption (OR, 5.8; 95% CI, 1.1-30) among white women. All results were null for MTHFR genotypes. CONCLUSION FVL and AGT variant genotypes were associated specifically with hemorrhage-related PTDs.

Challenges and solutions for gene identification in the presence of familial locus heterogeneity

Next-generation sequencing (NGS) of exomes and genomes has accelerated the identification of genes involved in Mendelian phenotypes. However, many NGS studies fall short of identifying causal variants, with estimates for success rates as low as 25% for uncovering the pathological variant underlying disease etiology. An important reason for such failures is familial locus heterogeneity, where within a single pedigree causal variants in two or more genes underlie Mendelian trait etiology. As examples of intra- and inter-sibship familial locus heterogeneity, we present 10 consanguineous Pakistani families segregating hearing impairment due to homozygous variants in two different hearing impairment genes and a European-American pedigree in which hearing impairment is caused by four variants in three different genes. We have identified 41 additional pedigrees with syndromic and nonsyndromic hearing impairment for which a single previously reported hearing impairment gene has been identified but only segregates with the phenotype in a subset of affected pedigree members. We estimate that locus heterogeneity occurs in 15.3% (95% confidence interval: 11.9%, 19.9%) of the families in our collection. We demonstrate novel approaches to apply linkage analysis and homozygosity mapping (for autosomal recessive consanguineous pedigrees), which can be used to detect locus heterogeneity using either NGS or SNP array data. Results from linkage analysis and homozygosity mapping can also be used to group sibships or individuals most likely to be segregating the same causal variants and thereby increase the success rate of gene identification.