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Point and octapeptide repeat (24 bp) insertional mutations in the prion protein gene (PRNP) cause a dominantly transmitted dementia, associated with spongiform degeneration of the brain, astrocytic gliosis and neuronal loss due to cell accumulation of mutated protease resistant prion protein.1 The octapeptide repeat region lies between codon 51 and 91, and comprises a nonapeptide followed by a tandem repeat containing four copies of an octapeptide. The normal tandem length in healthy individuals is five repeats R1‐R2‐R2‐R3‐R4, but mutations can contain up to nine additional extra repeats.2
An extra repeat number has been related to anticipated age at onset in affected subjects.2 When genetic testing fails to disclose evidence of parental transmission in a dominant disease, a negative family history in patients carrying extra repeats in PRNP could be related either to non‐paternity, to variability in mutation penetrance or to de novo mutations. Even in the absence of positive genetic tests in mutation carrier parents, Goldfarb et al hypothesised that genetic mechanism generating extra repeats might be unequal crossover.2 Some insight into this genetic mechanism comes from the de novo meiotic insertional extra repeat mutation in PRNP we detected in a patient whose parents had a normal phenotype and a wild‐type sequence in the same gene. To our knowledge, this is the first time this condition has been described.
We report the case of a 20‐year‐old patient with a negative family history and a 168 bp insertional PRNP mutation leading to seven extra repeats. A blood sample was obtained from the patient after informed consent and DNA was extracted from leucocytes by standard procedures. A molecular diagnosis was made by size discrimination of a PCR amplicon on agarose gels. The mutation was characterised by sequencing of both alleles. To sequence the two bands separately, they were excised and purified. The resulting DNA samples were sequenced with a Big Dye terminator sequencing kit and an ABI3100 genetic analyser (Applied Biosystem, Applera Italia, Monza, Italy). Coding exon of the PRNP were amplified using the published primers PP3 and PP8.1PRNP sequence analysis disclosed seven additional octapeptide coding repeats R1‐R2‐R2‐R3‐R2‐R2‐R3g‐R2‐R2‐R2‐R3‐R4, to our knowledge, in a fashion never described before in the Italian population.
The patient's clinical course followed the typical progression of Creutzfeldt–Jakob disease (CJD) due to extra octapeptides in PRNP, and was characterised by an initial intellectual decline starting from age 18 years with behavioural changes mainly characterised by apathy, irritability and isolation and rapidly progressing towards severe mental deterioration. At age 21 years, the patient was disoriented, interrupted his studies and showed severe depression and introversion. Psychiatric manifestations such as psychosis and bipolar disorder were also ascertained at age 26 years. In the ensuing years the disease developed slowly until age 28 years when neuropsychological tests showed severe intellectual decline with disorientation, memory impairment, difficulty in concentration and calculation, and right and left hand confusion. Motor symptoms consisted mainly of severe apraxia of voluntary movements and cerebellar ataxia, more severe in the lower than in the upper limbs, making tandem walking impossible. At this time, MRI showed mild but widespread cortical brain atrophy, with no evidence of hyperintensity in T2 weighted images.
Coherent to neuropsychological tests indicating scarce advancement of dementia in 2 years, [fluorine‐18]‐fluoro‐2‐deoxy‐D‐glucose (FDG) positron emission tomography (PET) scanning performed at ages 27 and 28 years (fig 1C1C)) revealed a non‐progressive pattern of cortical brain hypomethabolism, as described previously,3 which in our patient was confined to posterior areas. In agreement with these cases and in addition to the role of the extra repeat number, the polymorphism homozygous for the Met/Met at codon 129 of PRNP possibly contributed to the patient's relatively anticipated age at onset.3 Both parents, whose blood samples were collected for research purposes after informed consent, failed to show neurological or psychiatric symptoms at ages 60 and 55 years, however, and had a wild‐type sequence of PRNP (fig 1A1A).). Paternity was confirmed by variable number tandem repeat analysis. In an attempt to identify useful markers that could predict the parental origin of the specific allele with the insertional mutation in the PRNP gene, we sequenced the 1086 base pair near the 5′ end of the gene. We identified and examined in each family member the inheritance pattern of four, already described, single nucleotide polymorphisms (SNPs; rs6076717, rs6084835, rs6037933, rs11087653) and another common coding SNP (E219K). Unfortunately, genotyping of these four SNPs in DNA from the proband and parents failed to show any difference, each family member sharing identical genotypes.
This is, to our knowledge, the first reported genetic condition generating a de novo extra repeat insertional mutation in PRNP from unaffected parents both carrying a wild‐type PRNP sequence, and is the first report of such a mutation in a population of Italian origin.4,5 Seven extra repeat insertional mutations in PRNP have so far been described in only a few families4,5 to our knowledge, none originating as expanding from a wild‐type repeat sequence from one of the parents. This potential genetic mechanism was nevertheless proposed by Goldfarb et al, although they lacked evidence in genetically tested parents. Although a neuropathological assessment would definitely confirm the diagnosis, the case we report here lends support to the theory of Goldfarb et al of unequal meiotic crossovers generating additional extra repeat insertions in the PRNP gene of the offspring (fig 1B1B).). Our observation supports the hypothesis that a relatively large number of extra repeats (ie, seven) may generate from multiple crossovers in the same subject, thus causing a seven (or more) extra repeat mutation in offspring from unaffected parents (fig 1B1B).). The biological factors potentially influencing such a mechanism await further study. Apart from providing mechanistic clues on the genetic transmission of familial Creutzfeldt–Jakob disease in subjects with early dementia and a negative family history, possible de novo mutations should be sought in families with sporadic cases by genetic testing, thus requiring appropriate genetic counselling procedures.
Competing interests: None.