Literature

Literature = = Article 1 Overview The primary research article " Intragenic Deletions in ATP7B as an Unusual Molecular Genetics Mechanism of Wilson’s Disease Pathogenesis" sequenced the DNA of 1420 Wilson's disease patients, out of which 142 patients' DNA sequencing was unable to detect the ATP7B mutations. The aim of this was to study the unusual mutations in the WD patients. The 142 samples were examined and sequenced using Multiplex ligation-dependent probe amplification (MLPA).

What is MLPA? Multiplex ligation-dependent probe amplification (MLPA) is considered to be important for improving the detection of mutations in WD patients which is the basis for early diagnosis and predictive testing of at-risk relatives (Todorov, 2016). MLPA is a multiplex PCR that helps in the detection of mutations that were missed using routine sequencing.

MLPA Steps The MLPA reaction is very complicated, but the steps are as follows. In MLPA, it is not the target sequence that is amplified, but the complementary of the target sequence. The probes that are added to the samples are amplified and quantified.

The first step in MLPA is the denaturation of the DNA. The denatured DNA is kept overnight with the MLPA probes. Each probe consists of two separate oligonucleotides, but they both contain the PCR primer sequencing. Later the probes hybridize to adjacent sites of the target sequence. The hybridized probe oligonucleotides are ligated, which is followed by amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair (Schoten, 2002).

Result  Figure 2: Image is from the article Intragenic Deletions in ATP7B as an Unusual Molecular Genetics Mechanism of Wilson’s Disease Pathogenesis. Obtained by the permission of the author. <span style="background-color: #ffffff; font-family: &#39;Times New Roman&#39;; font-size: 12pt; vertical-align: baseline;">In this image B1 C1 and D1 are the exons that are amplified using MLPA. In B2, C2, and D2 the blue boxes represent the detection of the deleted genes. B3, C3, and D3 is the electrographic representation of the genes, in which the flancking regions are the representation of the deleted segments.


 * <span style="background-color: #ffffff; font-family: &#39;Times New Roman&#39;; font-size: 12pt; vertical-align: baseline;">New Mutations **

<span style="font-family: &#39;Times New Roman&#39;; font-size: 12pt; vertical-align: baseline;"> In the cohort of 1420 WD patients in this study the mutation detection rate obtained from routine sequencing was 90%. With the help of MLPA and selective amplification seven undetected mutations were identified in three different families. Therefore MLPA is considered to be better in detecting WD mutation, and is also considered to be a fast and inexpensive method. <span style="font-family: &#39;Times New Roman&#39;; font-size: 12pt; vertical-align: baseline;">

<span style="font-family: Times New Roman,Times,serif; font-size: 11.7px;">Figure 3: Image is from the article Intragenic Deletions in ATP7B as an Unusual Molecular Genetics Mechanism of Wilson’s Disease Pathogenesis. Obtained by the permission of the author.

<span style="font-family: &#39;Times New Roman&#39;; font-size: 12pt; vertical-align: baseline;">This table <span style="font-family: &#39;Times New Roman&#39;; font-size: 16px;">shows the seven new identified mutation in WD patients. These mutations were detected with the help of MLPA. <span style="font-family: &#39;Times New Roman&#39;; font-size: 12pt; vertical-align: baseline;">

<span style="font-family: Times New Roman,Times,serif; font-size: 120%;">Article 2 <span style="background-color: #ffffff; font-family: &#39;Times New Roman&#39;; font-size: 12pt; vertical-align: baseline;">Wilson’s disease can be treated by replacing diseased hepatocytes (liver cells) with healthy hepatocytes which is a cell therapy approach. The cells need the ATP7B-dependent copper transport to extract the excess copper through bile. This transport is deficient in WD leading to the copper accumulation in liver and brain. Replacing diseased hepatocytes with healthy stem cell derived liver cells or, immature hepatocytes in the liver has raised the possibility of successful and permanent cure of WD. <span style="background-color: #ffffff; font-family: &#39;Times New Roman&#39;; font-size: 12pt; vertical-align: baseline;">Suitable numbers of healthy cells are populated in a diseased liver according to the amount of damage that is done to the liver. The healthy hepatocytes must be placed in the parenchyma and restore the liver and the bile excretory system. This restoration is important for the proper excretion of the excess copper in the liver. In an experiment done on a rat's liver, its diseased cells were replaced by the healthy hepatocytes. This resulted in the restoration of the liver, and bile excretory system, and excess copper was excreted successfully. After this experiment the rats were monitored for their life span. These rats not only restored the normal function of the liver, but were also able to maintain the healthy liver activity throughout their lives (Gupta, 2014). <span style="background-color: #ffffff; font-family: &#39;Times New Roman&#39;; font-size: 12pt; vertical-align: baseline;">Although this study mostly talks about a treatment experimented on rats, but these developments are the road maps of successful cell and gene therapy to provide a permanent cure for Wilson's disease.

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