Coexistence of Unverricht-Lundborg disease and congenital deafness: Molecular resolution of a complex comorbidity


Address correspondence to Associate Prof. Dragoslav Sokić, Ph.D., Institute of Neurology, CCS, Dr. Subotića 6, 11000 Belgrade, Serbia. E-mail: dsokic@sezampro.yu


Purpose:  We report on genetic analysis of a complex condition in a Serbian family of four siblings, wherein two had progressive myoclonic epilepsy (PME) and congenital deafness (CD), one had isolated congenital deafness (ICD), and one was healthy.

Methods and Results:  Molecular diagnosis performed by Southern blotting confirmed Unverricht-Lundborg disease in the available sibling with PME/CD. In the sibling with ICD (heterozygote for expansion mutation in CSTB) we demonstrated recombination event between the D21S2040 marker and the CSTB gene and identified c.207delC (p.T70Xfs) mutation in the fourth exon of the transmembrane protease, serine-3 (TMPRSS3) gene (maps in close proximity to CSTB), responsible for nonsyndromic deafness in the sibling with PME/CD as well.

Discussion:  To the best of our knowledge this is the first genetic confirmation of the coexistence of these two mutations.

Unverricht-Lundborg disease (ULD) (EPM1, OMIM 254800), the most common of progressive myoclonic epilepsies (PMEs), is an autosomal recessive disorder caused by mutations in the cystatin B (CSTB) gene located on chromosome 21q22.3 (Pennacchio et al., 1996). Most patients with ULD carry a large dodecamer repeat expansion (40–80) in the promoter region of the CSTB(Lafrenière et al., 1997;Lalioti et al.,1997; Virtaneva et al., 1997), whereas normal alleles contain two to three copies.

A decade ago, it was found that mutations in two loci (DFNB8 /10) (Bonne-Tamir et al., 1996; Veske et al., 1996), mapping in proximity to the CSTB gene, were responsible for nonsyndromic recessive deafness. Mutations in the gene encoding a transmembrane serine protease, TMPRSS3, were found to be responsible for both phenotypes (Scott et al., 2001). Frequency of the TMPRSS3 mutations was estimated at 0.38% in the general Caucasian population (Wattenhofer et al., 2002).

In 1937 Latham and Munro reported an apparently recessive condition in a family of consanguineous parents in which five of eight children had a complex pattern consisting of PME and congenital hearing loss (Latham & Munro, 1937). A Lebanese pedigree with symptoms resembling those described by Latham and Munro but with distinct features was described recently (Megarbane et al., 1999). Here we present a Serbian family with the same phenotype wherein we assumed concurrence of responsible mutations in the CSTB and the TMPRSS3 genes.


Three of four siblings of healthy parents were born deaf but were otherwise normal. Spontaneous, action, and stimulus sensitive myoclonus appeared in individuals II-1 and II-3 in the 12th to 13th year of life (Fig. 1). Both had generalized tonic–clonic seizures (GTCS) at the early beginning of the disease that ceased with antiepileptic treatment. Because of the progressive course of axial and multifocal myoclonus over the years, resistant to treatment with valproic acid, clonazepam, and piracetam, patients with these diseases became confined to bed after 16 and 30 years from the disease onset, respectively. A 31-year-old individual II-1 died of complications secondary to a leg fracture. Female sibling II-3 (47 years) has been bedridden for 4 years because of severe jerking. The individual II-2 had congenital deafness but was otherwise healthy. We found neither obvious intellectual deterioration in all siblings nor data about consanguinity and neuropsychiatric disorders in four generations of their predecessors. This study was approved by the Ethical Committee of Clinical Center of Serbia. Blood samples of II-2, II-3, II-4, and their father (I-1) were obtained after written informed consent was obtained.

Figure 1.

 Haplotype analysis. Alleles are shown according to their sizes in base pairs. Sibling II-2 carries recombinant chromosome.

Genomic DNA was extracted from peripheral blood cells using standard procedures. Polymerase chain reaction (PCR) amplification of the promoter region of the CSTB gene was performed using primers described by Virtaneva et al. (1997). PCR fragments were Southern blotted and hybridized using standard method with (CCCCGCCCCGCG)2 oligonucleotide probe. The sizes of alleles in normal range were determined on 4% silver-stained denaturing polyacrylamide gel along with sequenced allelic ladder.

Seven markers flanking CSTB: D21S1890-D21S1885-D21S2040-CSTB-D21S1259-D21S1912-PFKL-D21S171 and one intragenic variant in the CSTB 3′ UTR (A2575G) were studied using haplotype analysis. PCR amplification was performed as previously described (Moulard et al., 2002). PCR products were run on 4% silver-stained denaturing PAGE along with a sequenced allelic ladder. Exons 1–13 of the TMPRSS3 gene and flanking sequences were amplified by PCR using primers reported by Scott et al. (2001). The amplified fragments were purified and directly sequenced.


Southern blot analysis revealed two expanded dodecamer repeats (54 and 50) in the promoter region of the CSTB in individual II-3, confirming the diagnosis of ULD, whereas the individual II-2 carried heterozygous expansion with 52 repeats. Individual II-4 had a number of repeats in the normal range. The father of the children was a heterozygous carrier with 50 repeats, indicating individual transmission instability in this family.

Haplotypes were manually reconstructed. Because the mother’s sample was not available, her haplotype was deduced. Haplotype analysis showed that in the individual II-2, the recombination event occurred between D21S2040 and the CSTB gene (Fig. 1), which represents a strong indication that the homozygous mutation in the TMPRSS3 could be responsible for deafness in this family. Subsequent sequencing analysis revealed that II-2 and II-3 were homozygotes for a deletion of one nucleotide in exon four (c.207delC, p.T70Xfs) of TMPRSS3, individual II-4 carries no mutation, and the parent was heterozygote for the same mutation (Fig. 2).

Figure 2.

 Sequencing analysis: (A) wt sequence detected in individual II-4; (B) c.del207C in TMPRSS3 gene detected in individuals II-2 and II-3; (C) heterozygous mutation in individual I-1.


Genetic analysis in this family with PME and innate deafness demonstrates the coexistence of homozygous expansion mutation in CSTB and deletion of one nucleotide in TMPRSS3, loci that map in proximity on 21q22.3. To our best knowledge the present report is the first molecular confirmation of the coexistence of the two mutations mentioned previously.

Because of its proximity to the CSTB gene, the TMPRSS3gene was the first gene of choice for nonsyndromic deafness in our study. There are numerous loci accounting for nonsyndromic deafness, therefore, we performed haplotype analysis in order to analyze if the mutation in the TMPRSS3 gene could be responsible for deafness in this family, as it was in other pedigrees of various ethnic origins (Ben-Yosef et al., 2001; Masmoudi et al., 2001). However, because two affected individuals in the analyzed pedigree have PME and CD and one individual has isolated inborn deafness, one could exclude coexistence of CSTB and TMPRSS3 mutations due to assumed joint transmission. Namely, if there is coexistence of two mutations in genes mapping to 21q22.3, individual with nonsyndromic deafness should carry homozygous expansion in the CSTB. However, haplotype analysis revealed the occurrence of the recombination event that could explain disjoint of the two mutations. This was the strong indication that the TMPRSS3 mutation is the cause of deafness, as it was shown by further sequencing analysis.

The fact that both mentioned mutations are on the same haplotype suggests that parents in this pedigree might have been distant relatives (we obtained data for only four generations of this family), especially given that they derived from the same Serbian village with a current population of approximately 200 inhabitants. Furthermore, other analyzed Serbian patients with ULD carry dodecamer repeat expansion on the same haplotype as family under study, but without mutation in the TMPRSS3 gene (Kecmanović M, Ristić AJ, Sokić D, Keckarević-Marković M, Vojvodić N, Ercegovac M, Janković S, Keckarević D, Savić-Pavićević D, Romac S, unpublished data). We assume that mutation in the TMPRSS3 gene originated independently on the chromosome that already carried expansion in CSTB and transmitted without interference through generations because of its recessive nature.

This is one of the reports of the phenotype consisting of PME and CD. Latham and Munro (1937) first described five of eight siblings born deaf with the onset of slowly progressive myoclonus at the age of 10–12 years leading to bed-confinement after 12–40 years of disease, irregular GTCS, and “no intellectual deterioration.”Megarbane et al. (1999) reported consanguineous pedigree with sensorineuronal deafness and myoclonic epilepsy. Nevertheless, disease course, late onset of myoclonic seizures (approximately age 30 years) triggered mainly with fever, macular pigmentation, and stroke-like episodes make distinctive differences between the family described by Latham and Munro and the one described in this article. Remarkable clinical similarities of affected individuals in one Serbian family and patients in previously described Oxfordshire’s pedigree (Latham & Munro, 1937) might hold a clue to a single explanation in those two. However, lack of proof that these families are related makes this association almost certainly coincidental. Yet, genetically proven coexistence of the two molecularly distinct disorders in Serbian family fits to Latham and Munro’s hypothesis that “myoclonus and deaf-mutism are two separate entities caused by different single recessive genes” in their pedigree.

This report shows how molecular tools can be utilized to dissect complex phenotypes in an individual, that is, it emphasizes the importance of searching for additional genetic explanation when the clinical features are not pertinent for a single genetic diagnosis.


This work was funded by the Ministry of Science, Republic of Serbia (project no. 145057-Đ and 143013).

We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines.

Disclosure: None of the authors has any conflict of interest to disclose.