Agnieszka Pollak

M34T and V37I mutations in GJB2 associated hearing impairment: Evidence for pathogenicity and reduced penetrance

Despite research the role of the M34T and V37I variants of GJB2 in causing hearing impairment (HI) remains controversial. Our purpose was to test a hypothesis that M34T and V37I are pathogenic but have distinct features resulting in a reduced penetrance. We screened for known GJB2/GJB6 mutations 233 Polish consecutive unrelated subjects with non‐syndromic, sensorineural HI who were previously found to carry 35delG mutation on one chromosome. The most frequent mutations were also analyzed in ∼1,000 controls. We found that M34T and V37I were significantly (P ≪ 10−6) overrepresented among patients, but their penetrance was estimated as 1/10 relative to mutations of undisputed pathogenicity. This finding apparently could not be explained by low degree of HI associated with M34T and V37I since another mutation causing comparably mild HI (L90P) did not have reduced penetrance. Subsequent analyses showed that the patients with M34T/35delG and V37I/35delG had significantly later onset of HI than patients with other genotypes (P < 10−6) including the L90P/35delG (P = 0.006). Also, among these patients (but not others) a strong correlation between the degree of HI and its duration was found (r = 0.79, P < 10−5). We tentatively suggest that M34T and V37I might cause mild HI characterized by relatively late onset and progression.

Does p.Q247X in TRIM63 Cause Human Hypertrophic Cardiomyopathy

Rationale: Variants in TRIM63, including a nonsense mutation (p.Q247X), have been suggested recently to cause hypertrophic cardiomyopathy. Objective: To verify pathogenicity of TRIM63 p.Q247X detected by whole-exome sequencing in a symptomless professional sports player seeking medical advice because of a prolonged QT interval found during a routine check-up. Methods and Results: Clinical studies were performed in the proband and his mother, who also carried TRIM63 p.Q247X. No evidence of hypertrophic cardiomyopathy was found in either person. Conclusions: The p.Q247X variant in TRIM63 is not likely to be a highly penetrant variant causing hypertrophic cardiomyopathy.

Molecular background of polyendocrinopathy-candidiasis-ectodermal dystrophy syndrome in a Polish population: novel AIRE mutations and an estimate of disease prevalence.

Autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) is an autosomal‐recessive autoimmune disease caused by autoimmune regulator gene mutations. The aim of this study was to examine the mutation profile of Polish APECED patients, determine the carrier rate of the most frequent mutation(s) and estimate disease prevalence. While studying 14 unrelated patients, we identified three novel mutations (c.1A>T, affecting the start codon; [IVS1 + 1G>C; IVS1 + 5delG], a complex mutation affecting splice site; c. 908G>C, p.R303P, a missense mutation in plant homeodomain (PHD) and three previously reported mutations (c.769C>T, p.R257X; c.967_979del13bp, C322fsX372; c.931delT, p.C311fsX376). Eleven patients had mutations on both chromosomes, whereas in three patients only a single alteration with proven or likely pathogenic effect was detected. The most frequent was the p.R257X mutation (71% of chromosomes); its carriage rate was assessed in the background population. Analysis of 2008 samples showed eight heterozygotes, indicating the frequency of 0.40% (1:250) and the disease prevalence – 1:129,000 (95% confidence interval: 1:555,000 to 1:30,000). Comparison with an epidemiological estimate (1:619,000, derived for women) suggested that in Poland, APECED is underdiagnosed. Among the patients, no genotype/phenotype correlations were found, but we noted that women had earlier onset of hypoparathyroidism (p < 0.02) and were younger at diagnosis (p < 0.05) than men.

Mutation analysis of mitochondrial 12S rRNA gene in Polish patients with non-syndromic and aminoglycoside-induced hearing loss.

Mutations in mitochondrial DNA have been reported as associated with non-syndromic and aminoglycoside-induced hearing loss. In the present study, we have performed mutational screening of entire 12S rRNA gene in 250 unrelated patients with non-syndromic and aminoglycoside-induced hearing loss. Twenty-one different homoplasmic sequence variants were identified, including eight common polymorphisms, one deafness-associated mutation m.1555 A>G and three putatively pathogenic variants: m.669 T>C, m.827 A>G, m.961 delT+C(n)ins. The incidence of m.1555 A>G was estimated for 3.6% (9/250); however, where aminoglycoside exposure was taken as a risk factor, the frequency was 5.5% (7/128). Substitution m.669 T>C was identified only in patients with hearing impairment and episode of aminoglycoside exposure, which may suggest that such additional risk factors must appear to induce clinical phenotype. Moreover, two 12S rRNA sequence variants: m.988 G>A and m.1453 A>G, localized at conserved sites and affected RNA secondary structure, may be new candidates for non-syndromic and aminoglycoside-induced hearing loss associated mutations.

GJB2 and hearing impairment: promoter defects do not explain the excess of monoallelic mutations

The report by Matos et al 1 brings up an issue of an excessive frequency of patients with hearing impairment (HI) in whom only monoallelic GJB2 mutations are identified by commonly used testing strategies. Interestingly, Matos et al suggest that these patients could harbour mutations in promoter of GJB2 which therefore should be included in the routine screening.1 This conclusion was supported by a novel sequence variant (−3438T→C) affecting GJB2 promoter function and found in a Portuguese patient in trans with a coding region mutation.1 The relatively high frequency of genotypes with monoallelic GJB2 mutations, such as the 35delG mutation, among the patients has indeed been noted previously2 3 although the exact size of this effect in relation to the generally high frequency of 35delG in Caucasians (carrier rates reaching 4%)4 has not been estimated. Although screening of promoter regions is clearly an attractive option in patients with monoallelic GJB2 defects, before it can be included in routine testing it should be shown that the −3438T→C or similar alteration(s) do indeed occur with an appreciable frequency. Given the report of Matos et al ,1 we were interested to find out: (1) whether there is an excess of monoallelic GJB2 mutations among patients with HI from our centre and if so, what is its magnitude, and (2) what is the frequency of GJB2 promoter mutations such as −3438T→C among such patients. Owing to the high frequency …

Spondyloepimetaphyseal dysplasia with neurodegeneration associated with AIFM1 mutation – a novel phenotype of the mitochondrial disease

In 1999, based on a single family, spondyloepimetaphyseal dysplasia (SEMD) with mental retardation (MR) was described as a novel syndrome with probably X‐linked recessive inheritance and unknown molecular defect (MIM 300232). Our purpose was to search for the causative defect in the originally described family and in an independently ascertained second family. All patients had slowly progressive neurodegeneration with central and peripheral involvement and identical skeletal dysplasia. Whole exome sequencing performed in two subjects showed a single plausible candidate – the p.Asp237Gly variant in AIFM1 (chr. Xq26.1). The p.Asp237Gly segregated with disease as indicated by linkage analysis [maximum logarithm of odds score (LOD) score at theta 0 for the two families was 3.359]. This variant had not been previously reported and it was predicted to be pathogenic by Polyphen2, SIFT, MutationTaster and Mutation Assessor. AIFM1 encodes mitochondria associated apoptosis‐inducing factor. The AIFM1 gene has been linked with COXPD6 encephalomyopathy (MIM 300816), Cowchock syndrome (MIM 310490) and X‐linked deafness with neuropathy (DFNX5, MIM 300614), none of which are similar to SEMD‐MR. Our results place SEMD as the third instance of a skeletal phenotype associated with a mitochondrial disease (the others being EVEN‐PLUS syndrome caused by mutations of HSPA9 and CODAS syndrome due to LONP1 mutations).

Evidence for troponin C (TNNC1) as a gene for autosomal recessive restrictive cardiomyopathy with fatal outcome in infancy.

Restrictive cardiomyopathy is a rare form of pediatric cardiac disease, for which the known genes include MYH7, TNNT2, TNNI3, ACTC1, and DES. We describe a pediatric proband with fatal restrictive cardiomyopathy associated with septal hypertrophy and compound heterozygosity for TNNC1 mutations (NM_003280: p.A8V [c.C23T] and p.D145E [c.C435A]). This association between restrictive cardiomyopathy and TNNC1 mutations was strengthened by prospective observations on the second pregnancy in the family which revealed, in the presence of the same TNNC1 genotype, prenatally diagnosed hypertrophic cardiomyopathy which evolved into restrictive cardiomyopathy, heart failure and death at the age of 9 months. Contrary to previous reports, family and population analyses showed that each of the TNNC1 variants was not pathogenic when present alone. Our results (i) confirm that genetic backgrounds of hypertrophic cardiomyopathy and restrictive cardiomyopathy overlap and (ii) indicate that TNNC1 is a likely novel gene for autosomal recessive restrictive cardiomyopathy.

Novel and De Novo Mutations Extend Association of POU3F4 with Distinct Clinical and Radiological Phenotype of Hearing Loss.

POU3F4 mutations (DFNX2) are the most prevalent among non-syndromic X-linked hearing loss (HL) identified to date. Clinical manifestations of DFNX2 usually comprise congenital HL either sensorineural or mixed, a tendency towards perilymphatic gusher during otologic surgery and temporal bone malformations. The aim of the present study was to screen for POU3F4 mutations in a group of 30 subjects with a suggestive clinical phenotype as well as a group (N = 1671–2018) of unselected hearing loss patients. We also planned to analyze audiological and radiological features in patients with HL caused by POU3F4 defects. The molecular techniques used to detect POU3F4 mutations included whole exome sequencing (WES), Sanger sequencing and real-time polymerase chain reaction. Hearing status was assessed with pure-tone audiometry and auditory brainstem response. Computer tomography scans were evaluated to define the pattern of structural changes in the temporal bones. Six novel (p.Gln27*, p.Glu187*, p.Leu217*, p.Gln275*, p.Gln306*, p.Val324Asp) and two known (p.Ala116fs141*, p.Leu208*) POU3F4 mutations were detected in the studied cohort. All probands with POU3F4 defects suffered from bilateral, prelingual, severe to profound HL. Morphological changes of the temporal bone in these patients presented a similar pattern, including malformations of the internal auditory canal, vestibular aqueduct, modiolus and vestibule. Despite different localization in the POU3F4 gene all mutations severely impair the protein structure affecting at least one functional POU3F4 domain, and results in similar and severe clinical manifestations. Sequencing of the entire POU3F4 gene is recommended in patients with characteristic temporal bone malformations. Results of POU3F4 mutation testing are important not only for a proper genetic counseling, but also for adequate preparation and conduction of a surgical procedure.

Evidence Against RAB40AL Being the Locus for Martin–Probst X‐Linked Deafness–Intellectual Disability Syndrome

RAB40AL has been reported as the locus for Martin–Probst syndrome (MPS), an X‐linked deafness–intellectual disability syndrome. The report was based on segregation of a missense change p.D59G with the disease in a single family and in vitro localization studies. We found the p.D59G variant by whole‐exome sequencing in two patients; however, the diagnosis of MPS was excluded in both cases. Furthermore, screening of control DNA samples (n = 810) from a general Polish population, using allele‐specific PCR and direct DNA sequencing for verification, identified p.D59G in 8/405 males and 12/405 females. High prevalence of the p.D59G variant (2.47%) is typical for a common genetic variation observed in asymptomatic individuals. Our data question the role of RAB40AL mutation as a disease‐causing change and the involvement of RAB40AL in MPS. Considering an increasing use of next‐generation sequencing in the clinical setting, our finding is of practical diagnostic importance.

Postlingual Hearing Loss as a Mitochondrial 3243A>G Mutation Phenotype

Background The prevalence of isolated hearing loss (HL) associated with the m.3243A>G mutation is unknown. The aim of this study was to assess the frequency and heteroplasmy level of the m.3243A>G mutation in a large group of Polish patients with postlingual bilateral sensorineural HL of unidentified cause. Methodology/Principal Findings A molecular search was undertaken in the archival blood DNA of 1482 unrelated patients with isolated HL that had begun at ages between 5 and 40 years. Maternal relatives of the probands were subsequently investigated and all carriers underwent audiological tests. The m.3243A>G mutation was found in 16 of 1482 probands (an incidence of 1.08%) and 18 family members. Of these 34 individuals, hearing impairment was detected in 29 patients and the mean onset of HL was at 26 years. Some 42% of the identified m.3243A>G carriers did not develop multisystem symptomatology over the following 10 years. Mean heteroplasmy level of m.3243A>G was lowest in blood at a level of 14% and highest in urine at 58%. These values were independent of the manifested clinical severity of the disease. Conclusions A single m.3243A>G carrier can usually be found among each 100 individuals who have postlingual hearing loss of unknown cause. Urine samples are best for detecting the m.3243A>G mutation and diagnosing mitochondrially inherited hearing loss.