Patient details
The patient was born, after an uneventful pregnancy and delivery, weighing 3.4 kg. He was the younger of two siblings born to non-consanguineous Caucasian parents. His very early motor milestones were acquired age appropriately, and his overall development was normal until eight months of age, at which time he had a febrile fit and lost some of his acquired skills. When referred at the age of 15 months, his development was at an approximate age level of six to eight months. Seizures developed at four years of age, but these were well controlled with sodium valpro-ate and lamotrigine. They were a combination of tonic/clonic, 'star' seizures (tonic extension) and brief atonic attacks. By this time, global developmental delay was evident. He was first seen for a genetics review at the age of ten years. His gait was unsteady, with a tendency to fall frequently or bump into objects. He was able to walk short distances but longer distances required the use of a wheelchair. Although he was able to walk upstairs holding onto a rail, he descended stairs on his bottom. He spoke a few solitary words and could obey simple commands. He was not dysmorphic, but had a degree of brachycephaly. His head circumference was on the 75th centile, weight on the 98th centile and height on the 25th centile. He had relatively small genitalia, but no neurocutaneous stigmata.
The patient had a normal 46XY karyotype, with no evidence of a microdeletion at the Miller-Dieker locus on 17p13.3. His EEG at the age of six years was abnormal, with frequent showers of sharp waves or spikes throughout the recording. This was more obvious in the anterior brain regions, with some emphasis on the left side. A magnetic resonance imaging (MRI) brain scan at eight years of age revealed frontal, parietal, posterior temporal and occipital pachygyria, with maximal cortical thickening posteriorly. The degree of pachygyria was also milder anteriorly. Appearances were consistent with classical lissencephaly at the milder end of the spectrum, and a posterior to anterior severity consistent with the presence of a PAFAH1B1 (LIS1) gene mutation. In view of the highly unusual PAFAH1B1 gene insertion found in this patient, genomic DNA from this individual was also screened for DCX gene mutations, but none were found.
Microsatellite marker analysis was used to confirm that familial relationships were as stated at referral (data not shown).
Sequence analysis of the PAFAH1B1gene in the patient
PCR amplification and sequencing of the coding region of the PAFAH1B1 gene (exons 2 to 11) of the proband's DNA revealed a heterozygous 130 bp insertion within exon 2. This insertion was located within the 5' UTR, seven bp upstream of the translational initiation site (ATG) (between bases 560 and 562 of the reference sequence [accession number NM_000430]; Figure 1). The 130 bp insertion contained two non-templated bases (TT) at its 5' end and was accompanied by the deletion of a cytosine at position 561 of the PAFAH1B1 gene (accession number NM_000430). The mutation may therefore be described as g.-8delCins130. No other sequence alterations in any other exon or splice site of the PAFAH1B1 gene were identified. Sequence analysis of exon 2 of the PAFAH1B1 gene in both parents indicated only the presence of the wild-type sequence, consistent with the de novo occurrence of the mutation in the patient.
Origin of the inserted DNA sequence with homology to the mitochondrial genome
A BLAST search was performed to determine the origin of the inserted sequence. Perfect homology to the mitochondrial genome sequence (8479 to 8545 and 8775 to 8835; accession number NC_001807.4) was noted, as well as near-perfect homology to a NUMT sequence [13] at chromosome 1p36 (47659 to 47727 and 47955 to 48015; accession number NT_004350.19). The homology between the 130 bp inserted sequence and the mitochondrial genome/NUMT was not, however, contiguous; rather, two regions of mitochondrial DNA sequence homology (of length 67 bp and 61 bp, respectively) were noted, which were located 229 bp distant from each other in the mitochondrial genome/NUMT. The 67 bp and 61 bp sequences were both identical to the mitochondrial genome reference sequence, whereas, in the case of the sequence of the telomerically located chromosome 1-specific NUMT, the 67 bp fragment contained one mismatch (Figure 2).
Inspection of the sequence flanking the junction between the 67 bp and 61 bp fragments identified two short imperfect direct repeats, GAAGC and GGAGG, in the mitochondrial genome (8546 to 8550 and 8776 to 8780, respectively; accession number NC_001807.4) which could have mediated the loss of the 229 bp fragment through slipped mispairing. It should be noted that the equivalent sequences in the chromosome 1 NUMT are GAAGT and GGAGG (47728 to 47732 and 47956 to 47960, respectively; accession number NT_004350.19).
The inserted sequence contains portions of two mitochondrial genes, ATP8 (8367 to 8573; accession number NC_001807.4) and ATP6 (8528 to 9208; accession number NC_001807.4), but whether the inserted sequence was derived from the mitochondrial genome itself or from a reverse transcript of mRNA encoding the ATP6 and ATP8 mitochondrial genes cannot be ascertained from the DNA sequence involved.
Since the mitochondrial genome and chromosome 1-specific NUMT sequences differed from each other by only 1 bp over the 130 bp length of the insert, it was considered important to establish whether the patient's own mitochondrial genome and chromosome 1-specific NUMT sequences were identical to their respective published reference sequences. Oligonucleotide primers were therefore designed specifically to PCR amplify DNA fragments corresponding to the inserted sequence from either the mitochondrial genome or chromosome 1. PCR products from the mitochon-drial genome of both the patient and his parents were sequenced and found to be identical to (but not, of course, contiguous with) the sequence inserted into the PAFAH1B1 gene. Similarly, PCR/direct sequencing of the chromosome 1-specific NUMT from both the patient's parents confirmed sequence identity with the standard chromosome 1 reference sequence (ie 1 bp mismatch with respect to the PAFAH1B1 gene insertion). Attempts to PCR amplify the chromosome 1-specific NUMT sequence from the patient repeatedly failed to yield any PCR product, however. To confirm that the nuclear DNA from the patient was of good quality, PCR amplification of a 3.2 kb fragment containing the GH1 gene [14] was performed. Successful PCR amplification of this fragment from patient DNA (data not shown) indicated that the lack of PCR amplification of the chromosome 1-specific NUMT was not due to poor DNA quality. The reason why the patient's chromosome 1-specific NUMT was refractory to analysis remains unclear but is potentially interesting, given the possible involvement of this sequence in the PAFAH1B1 gene insertion. Although it remains the most likely scenario, in the absence of DNA sequence information from the patient's own chromosome 1-specific NUMT, we cannot unequivocally confirm that the 130 bp insertion originated from the mitochondrial genome sequence rather than from the NUMT.
In order to ascertain whether the patient possessed a deletion of chromosome 1p36 encompassing the chromosome 1-specific NUMT, an attempt was made to PCR amplify across the NUMT sequence. Combinations of different chromosome 1-specific primers (Table S2) were employed to amplify different DNA sequences between 43,267 and 53,538 (accession Number NT_004350.19) in both the patient and his parents. PCR products of the appropriate sizes were amplified from both parents and, on sequencing, were confirmed to match the chromosome 1-specific NUMT sequence. No PCR product from the chromosome 1-specific NUMT was obtained from the patient for any combination of primers used, however, suggesting that this region of chromosome 1 may have been homozygously deleted, rearranged or both.
Analysis of the inserted sequence and the site of insertion in the PAFAH1B1gene
The 130 bp sequence inserted into the 5' UTR of the PAFAH1B1 gene contains two out-of-frame ATGs that could, at least in principle, serve as alternative translational initiation codons. The sequences flanking these ATGs GTGTAAATGA (positions 263 to 272; Figure 3) and ATTTTTATGG (positions 344 to 353; Figure 3) -- do not match the Kozak consensus sequence (GCC(A/G)NNATGG), [15] however, unlike the wild-type sequence (GCCAAGATGG) in the PAFAH1B1 gene. This suggests that neither of these sites is likely to be able to play a role in translational initiation. The insertion occurs at nucleotide position -7 relative to the ATG, immediately adjacent to the 5' end of the Kozak consensus sequence.
Since the identified insertion lies within the 5' UTR of the PAFAH1B1 gene, it has the potential to have an impact on RNA secondary structure. Using RNAfold, the optimal secondary structure minimum free energy was determined to be -249.0 kcal/mol for the wild-type 5' UTR, whereas for the mutant 5' UTR (containing the insertion), this value was -299.6 kcal/mol. This suggests that the stability of the 5' UTR may not have been dramatically altered by the insertion; however, the predicted secondary structure of the mutant 5' UTR molecule was clearly very different from that of the wild-type 5' UTR (Figure S1).
Differences in pre-mRNA structure resulting from single bp substitutions have been reported to result in aberrant splicing [16]. Prediction of splice sites in the wild-type PAFAH1B1 sequence using NNSPLICE software [12] attributed the experimentally validated exon 2 acceptor (position 50; Figure 3) and donor (position 401; Figure 3) splice sites with potential splice site scores of 0.97 and 1.00, respectively. When the analysis was repeated for the mutant PAFAH1B1 sequence, an additional acceptor splice site was predicted (position 325, score 0.91; Figure 3) in addition to the wild-type splice sites. Without in vitro splicing analysis, however, it remains unclear whether this additional acceptor splice site would be functionally significant.
Mechanism of mutagenesis
No sequence homology was found between the site of insertion in the PAFAH1B1 gene and the mitochondrial genome that could have explained how the mitochondrial DNA fragment became integrated at this position. Two highly homologous regions, marked 1 and 2 in Figure 4, were, however, identified in the vicinity of the PAFAH1B1 gene. These regions could have led to a double-strand break through non-B slipped structure formation.