<p>L1 syndrome is an X-linked recessive disorder, characterised by congenital hydrocephalus, adducted thumbs, spastic paraplegia, agenesis of the corpus callosum and mental retardation. The disease is caused by mutations in the L1CAM gene, encoding a protein predominantly expressed in the nervous system. This protein has been implicated in a variety of processes including neuronal migration, neurite outgrowth and fasciculation, myelination, and long-term memory formation.</p><p>L1 syndrome was suspected at genetic counselling of a family with a boy suffering from congenital hydrocephalus and mental retardation. Complete sequencing of L1CAM, performed by an external laboratory, revealed a novel mutation in the family, including a boy, affected with L1 syndrome, his sister, his mother and his maternal grandmother.</p><p>To verify this mutation and to be able to detect mutations in the L1CAM gene locally in the future, a method for mutational analysis of this gene was set up using PCR optimisation and cycle sequencing.</p><p>Sequencing of L1CAM was then performed on DNA samples from the four family members and the mutation was verified. A point mutation (c.3458-1G>C) in the 5’ splice site of exon 26 was detected in all of them. This new, not previously described, mutation is supposed to cause a deletion of the 26th exon and a frameshift in the part of the protein encoded by exons 27 and 28. This means that almost the entire cytoplasmic domain of the protein would have a loss of function, explaining the symptoms affecting the boy.</p>

L1 syndrome is an X-linked recessive disorder, characterised by congenital hydrocephalus, adducted thumbs, spastic paraplegia, agenesis of the corpus callosum and mental retardation. The disease is caused by mutations in the L1CAM gene, encoding a protein predominantly expressed in the nervous system. This protein has been implicated in a variety of processes including neuronal migration, neurite outgrowth and fasciculation, myelination, and long-term memory formation. L1 syndrome was suspected at genetic counselling of a family with a boy suffering from congenital hydrocephalus and mental retardation. Complete sequencing of L1CAM, performed by an external laboratory, revealed a novel mutation in the family, including a boy, affected with L1 syndrome, his sister, his mother and his maternal grandmother. To verify this mutation and to be able to detect mutations in the L1CAM gene locally in the future, a method for mutational analysis of this gene was set up using PCR optimisation and cycle sequencing. Sequencing of L1CAM was then performed on DNA samples from the four family members and the mutation was verified. A point mutation (c.3458-1G&gt;C) in the 5’ splice site of exon 26 was detected in all of them. This new, not previously described, mutation is supposed to cause a deletion of the 26th exon and a frameshift in the part of the protein encoded by exons 27 and 28. This means that almost the entire cytoplasmic domain of the protein would have a loss of function, explaining the symptoms affecting the boy.

Health Tech company Dynamic Code AB provides a PCR test for determination of HLA DQ-genes connected to development of celiac disease. The PCR method is probe based and in real time and is at this time carried through on the, somewhat outdated, PCR instrument from Thermo Fisher/Applied Biosystems called 7300 Real-Time PCR System. The run time for this analysis on the instrument is 1 hour and 50 minutes. The Master Mix in use is TaqMan™ Gene Expression Master Mix, from the same manufacturer. Moving on to a more modern PCR instrument is a natural step for the company and is favourable in several regards, one of them being the run time that will be cut by 50 minutes, allowing for more samples to be analysed in the same amount of time. The objective is to move the HLA analysis to Thermo Fisher’s QuantStudio™ 6 and 7 Flex Systems and at the same time change the Master Mix to SolisFast® Probe qPCR Mix (Purple) from Solis BioDyne, in order to achieve better accuracy as this Master Mix is more compatible with the latter instrument, along with reducing reagent cost as it is less expensive. In order to find the optimal primer and probe concentration for each target included in the HLA analysis, their concentrations were varied and tested with the new Master Mix on the new instrument. PCR instrument transfer and Master Mix change was successful and validation experiments showed a 98,9% accuracy for the new method compared to the original method.