Nanna Dahl Rendtorff

A novel classification system to predict the pathogenic effects of CHD7 missense variants in CHARGE syndrome

CHARGE syndrome is characterized by the variable occurrence of multisensory impairment, congenital anomalies, and developmental delay, and is caused by heterozygous mutations in the CHD7 gene. Correct interpretation of CHD7 variants is essential for genetic counseling. This is particularly difficult for missense variants because most variants in the CHD7 gene are private and a functional assay is not yet available. We have therefore developed a novel classification system to predict the pathogenic effects of CHD7 missense variants that can be used in a diagnostic setting. Our classification system combines the results from two computational algorithms (PolyPhen‐2 and Align‐GVGD) and the prediction of a newly developed structural model of the chromo‐ and helicase domains of CHD7 with segregation and phenotypic data. The combination of different variables will lead to a more confident prediction of pathogenicity than was previously possible. We have used our system to classify 145 CHD7 missense variants. Our data show that pathogenic missense mutations are mainly present in the middle of the CHD7 gene, whereas benign variants are mainly clustered in the 5′ and 3′ regions. Finally, we show that CHD7 missense mutations are, in general, associated with a milder phenotype than truncating mutations. Hum Mutat 33:1251–1260, 2012.

Identification of p.A684V Missense Mutation in the WFS1 Gene as a Frequent Cause of Autosomal Dominant Optic Atrophy and Hearing Impairment

Optic atrophy (OA) and sensorineural hearing loss (SNHL) are key abnormalities in several syndromes, including the recessively inherited Wolfram syndrome, caused by mutations in WFS1. In contrast, the association of autosomal dominant OA and SNHL without other phenotypic abnormalities is rare, and almost exclusively attributed to mutations in the Optic Atrophy‐1 gene (OPA1), most commonly the p.R445H mutation. We present eight probands and their families from the US, Sweden, and UK with OA and SNHL, whom we analyzed for mutations in OPA1 and WFS1. Among these families, we found three heterozygous missense mutations in WFS1 segregating with OA and SNHL: p.A684V (six families), and two novel mutations, p.G780S and p.D797Y, all involving evolutionarily conserved amino acids and absent from 298 control chromosomes. Importantly, none of these families harbored the OPA1 p.R445H mutation. No mitochondrial DNA deletions were detected in muscle from one p.A684V patient analyzed. Finally, wolframin p.A684V mutant ectopically expressed in HEK cells showed reduced protein levels compared to wild‐type wolframin, strongly indicating that the mutation is disease‐causing. Our data support OA and SNHL as a phenotype caused by dominant mutations in WFS1 in these additional eight families. Importantly, our data provide the first evidence that a single, recurrent mutation in WFS1, p.A684V, may be a common cause of ADOA and SNHL, similar to the role played by the p.R445H mutation in OPA1. Our findings suggest that patients who are heterozygous for WFS1 missense mutations should be carefully clinically examined for OA and other manifestations of Wolfram syndrome.

A novel missense mutation in ACTG1 causes dominant deafness in a Norwegian DFNA20/26 family, but ACTG1 mutations are not frequent among families with hereditary hearing impairment

The γ-actin gene (ACTG1) encodes a major cytoskeletal protein of the sensory hair cells of the cochlea. Recently, mutations in ACTG1 were found to cause autosomal dominant, progressive, sensorineural hearing impairment linked to the DFNA20/26 locus on chromosome 17q25.3 in four American families and in one Dutch family. We report here the linkage of autosomal dominant, progressive, sensorineural hearing impairment in a large Norwegian family to the DFNA20/26 locus. Sequencing of ACTG1 identified a novel missense mutation (c.1109T>C; p.V370A) segregating with the hearing loss. Functional analysis in yeast showed that the p.V370A mutation restricts cell growth at elevated temperature or under hyperosmolar stress. Molecular modelling suggested that the p.V370A mutation modestly alters a site for protein–protein interaction in γ-actin and thereby modestly alters γ-actin-based cytoskeletal structures. Nineteen Norwegian and Danish families with autosomal, dominant hearing impairment were analyzed for mutations in ACTG1 by sequencing, but no disease-associated mutations were identified. Finally, a long-term follow-up of the hearing loss progression associated with the p.V370A mutation in ACTG1 is provided. The present study expands our understanding of the genotype–phenotype relationship of this deafness gene and provides a sensitive and simple functional assay for missense mutations in this gene, which may assist future molecular diagnosis of autosomal-dominant hearing impairment. Finally, the present results do not indicate that mutations in ACTG1 are a frequent cause of autosomal-dominant postlingual sensorineural hearing impairment in Norway nor Denmark.

Branchio-oto-renal syndrome: detection of EYA1 and SIX1 mutations in five out of six Danish families by combining linkage, MLPA and sequencing analyses

The branchio–oto–renal (BOR) syndrome is an autosomal-dominant disorder characterized by hearing loss, branchial and renal anomalies. BOR is genetically heterogeneous and caused by mutations in EYA1 (8q13.3), SIX1 (14q23.1), SIX5 (19q13.3) and in an unidentified gene on 1q31. We examined six Danish families with BOR syndrome by assessing linkage to BOR loci, by performing EYA1 multiplex ligation-dependent probe amplification (MLPA) analysis for deletions and duplications and by sequencing of EYA1, SIX1 and SIX5. We identified four EYA1 mutations (c.920delG, IVS10−1G>A, IVS12+4A>G and p.Y591X) and one SIX1 mutation (p.W122R), providing a molecular diagnosis in five out of the six families (83%). The present, yet preliminary, observation that renal and temporal bone malformations are less frequent in SIX1-related disease suggests a slightly different clinical profile compared to EYA1-related disease. Unidentified mutations impairing mRNA expression or further genetic heterogeneity may explain the lack of mutation finding in one family despite LOD score indications of EYA1 involvement.

Effects of human deafness γ-actin mutations (DFNA20/26)on actin function

Six point mutations in non-muscle γ-actin at the DFNA20/26 locus cause autosomal dominant nonsyndromic hearing loss. The molecular basis for the hearing loss is unknown. We have engineered each γ-actin mutation into yeast actin to investigate the effects of these mutations on actin function in vivo and in vitro. Cells expressing each of the mutant actins as the sole actin in the cell were viable. Four of the six mutant strains exhibited significant growth deficiencies in complete medium and an inability to grow on glycerol as the sole carbon source, implying a mitochondrial defect(s). These four strains exhibited abnormal mitochondrial morphology, although the mtDNA was retained. All of the mutant cells exhibited an abnormally high percentage of fragmented/non-polarized actin cables or randomly distributed actin patches. Five of the six mutants displayed strain-specific vacuole morphological abnormalities. Two of the purified mutant actins exhibited decreased thermal stability and increased rates of nucleotide exchange, indicative of increased protein flexibility. V370A actin alone polymerized abnormally. It aggregated in low ionic strength buffer and polymerized faster than wild-type actin, probably in part because of enhanced nucleation. Mixtures of wild-type and V370A actins displayed kinetic properties in proportion to the mole fraction of each actin in the mixture. No dominant effect of the mutant actin was observed. Our results suggest that a major factor in the deafness caused by these mutations is an altered ability of the actin filaments to be properly regulated by actin-binding proteins rather than an inability to polymerize.

A novel nonsense mutation in MYO6 is associated with progressive nonsyndromic hearing loss in a Danish DFNA22 family

Autosomal dominant inheritance is described in about 20% of all nonsyndromic hearing loss with currently 54 distinct loci (DFNA1‐54), and >20 different genes identified. Seven different unconventional myosin genes are involved in ten different types of syndromic and nonsyndromic hearing loss with different patterns of inheritance: MYO7A in DFNA11/DFNB2/USH1B, MYH9 in DFNA17, MYH14 in DFNA4, MYO6 in DFNA22/DFNB37, MYO3A in DFNB30, MYO1A in DFNA48, and MYO15A in DFNB3. Two missense mutations in MYO6 (p.C442Y and p.H246R) have been characterized in families of Italian and American Caucasian extraction with autosomal dominant hearing loss, respectively, and the latter was associated with cardiomyopathy in some patients. Three Pakistani families had homozygosity for three MYO6 mutations (c.36insT, p.R1166X, and p.E216V, respectively), and was in one instance associated with retinal degeneration. In the present study, we linked autosomal dominant hearing loss in a large Danish family to a 38.9 Mb interval overlapping with the DFNA22/DFNB37 locus on chromosome 6q13. A novel nonsense mutation in MYO6 exon 25 (c.2545C > T; p.R849X) was identified in the family. The mutation co‐segregated with the disease and the mutant allele is predicted to encode a truncated protein lacking the coiled‐coil and globular tail domains. These domains are hypothesized to be essential for targeting myosin VI to its cellular compartments. No other system was involved indicating nonsyndromic loss. In conclusion, a novel nonsense MYO6 mutation causes post‐lingual, slowly progressive autosomal dominant nonsyndromic moderate to severe hearing loss in a Danish family.

The coding region of TP53INP2, a gene expressed in the developing nervous system, is not altered in a family with autosomal recessive non-progressive infantile ataxia on chromosome 20q11-q13

The locus for autosomal recessive infantile cerebellar ataxia (CLA3 or SCAR6) has been mapped to chromosome 20q11‐q13 in a single Norwegian pedigree. We identified a relatively uncharacterised mouse gene Tp53inp2, and showed that its human orthologue mapped within this candidate interval. Tp53inp2 appears to encode a mammalian‐specific protein with homology to the two Tp53inp1 isoforms that respond to cellular stress and interact with p53. We show that Tp53inp2 expression is highly restricted during mouse embryogenesis, with strong expression in the developing brain and spinal cord, as well as in the sensory and motor neuron tracts of the peripheral nervous system. Given this expression pattern, the neurological phenotype of CLA3 and the chromosomal localisation of TP53INP2, we searched the coding region for mutations in samples from individuals from the CLA3 pedigree. Our failure to detect causative mutations suggests that alterations in the coding region of TP53INP2 are not responsible for ataxia in this family, although we cannot rule out changes in non‐coding elements of this gene. Developmental Dynamics 236:843–852, 2007.

The phenotypic spectrum of dystonia in Mohr–Tranebjaerg syndrome

Mohr–Tranebjaerg syndrome (MTS) is an X‐linked recessive disorder characterized by deafness and dystonia. However the phenotypic expression of dystonia has not been systematically defined. We report clinical, neurophysiological, and ophthalmological data on 6 subjects from 3 Australian kindreds, including 2 with novel mutations, together with a systematic review of the literature, in order to define the phenotypic expression of dystonia. Profound hearing impairment in affected males develops by infancy and precedes the development of dystonia, which varies in time of onset from the first to the sixth decades, with a peak in the second and third decades. Dystonia in MTS tends to be focal, segmental, or multifocal in distribution at onset, with a predilection for the upper body, variably involving the head, neck, and upper limbs. The majority of patients have progression or generalization of their dystonia regardless of age of onset. Within our 3 kindreds, we observed relative intrafamilial homogeneity but interfamilial variation. The median time to the development of moderate‐severely disabling dystonia in these subjects was 11 years. Associated features included progressive cognitive decline, pyramidal signs, and in 1 patient, gait freezing and postural instability. Optic atrophy and cortical visual impairment were both observed. We report for the first time a female patient who developed multiple disabling neurological complications of MTS. Our findings more clearly define and expand the phenotype of both the dystonia and other neurological features of MTS and have implications for the diagnosis and management of this condition.

Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting

Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss and mutations in the SLC26A4 gene. Extensive allelic heterogeneity, however, necessitates analysis of all exons and splice sites to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously identified by sequencing. Subsequently, a 384-well plate format was designed for up to three patient samples per run. Blinded HRM testing on these plates of patient samples collected over 1 year in a clinical diagnostic laboratory accurately detected all variants identified by sequencing. In conclusion, HRM with targeted sequencing is a reliable, simple, and cost-effective method for SLC26A4 mutation screening and detection.

GJB2 (Connexin-26) mutations are not frequent among hearing impaired patients in east Greenland

Objective: Investigate genetic causes of HI among the Inuit populations in the Arctic with a high prevalence of hearing impairment (HI). Design: A cross-sectional survey with population-based controls. Study sample: Forty-five patients, with sensorineural or mixed HI and an available blood sample for GJB2 sequencing from DNA, were selected from 166 East Greenlanders by specialist audiology examination, including pure-tone air and bone conduction audiometry from 125 Hz to 8000 Hz. Controls were 108 East- and 109 West-Greenlanders. Results: Forty-five patients with HI were included, 24 males and 21 females. Median age was 35 years (range: 5–76). The c.35delG allele frequency was 3.3%. One patient, homozygous for the c.35delG GJB2 mutation, had bilateral congenital profound HI. Another with mixed HI was heterozygous for the same mutation. Three were heterozygous for the p.V27I variant and one was heterozygous for the p.V153I variant. The frequency of the c.35delG mutation in the controls varied between 0.5% in West Greenland to 2.3% in East Greenland. Conclusion: The c.35delG GJB2 mutation occurs in Greenland with low frequency. We conclude the main causes behind the prevalence of HI in this population are chronic otitis media, noise traumas, and/or unidentified genetic causes.