Facioscapulohumeral muscular dystrophy (FSHD): Haplotype 4qA161 and PAS polymorphism (Polyadenylation signal) in pLAM region (FSHD1) and SMCHD1 gene (FSHD2).
Facio-scapulo-humeral muscular dystrophy (FSHD: FacioScapuloHumeral Dystrophy) is a process characterized by muscle weakness and atrophy. Its name is determined by the areas of the body that are most frequently affected: muscles of the face (facio-), muscles around the shoulders (scapulo-), and upper extremities (humeral-). The signs and symptoms of this dystrophy usually appear in adolescence, but both the moment of onset and the severity vary greatly; thus, mild cases can begin in advanced age, while severe cases, although rare, can present in infancy or early childhood.
Weakness of the facial and shoulders muscles are usually the first symptoms, and it tends to make it difficult to lift the mouth corners when smiling; the weakness of the eye muscles can prevent closing the eyes completely when sleeping, with the consequent dry eyes and the problems derived from it. The manifestation may affect one half of the face more than the other. The weakness of the shoulder muscles causes the scapulae to separate from the back (winged scapulae), preventing the arms from being raised above the head to launch an object. Muscle weakness worsens over the years and can spread to other areas of the body. In the lower extremities, when affected, it leads to foot drop, affecting gait and increasing the risk of falls. Muscle weakness in the hips and pelvis can make it difficult to climb stairs or walk long distances. Additionally, affected individuals may present with lordosis due to weak abdominal muscles. Additional signs and symptoms may include mild hearing loss and abnormalities affecting the retina. In rare cases, facioscapulo-humeral muscular dystrophy affects the heart muscle or the respiratory muscles.
Two types of facial-scapular-humeral muscular dystrophy have been described: type 1 (FSHD1) and type 2 (FSHD2). The two types have the same signs and symptoms and are distinguished by their genetic cause. Both types are due to changes in a region of the long arm of chromosome 4 called D4Z4 (macrosatellite), located near the end of this chromosome. This region, under normal conditions, has between 11 and 150 repeats, each of them 3,300 bp (3.3 kb) in length.
Normally, the entire D4Z4 region is hypermethylated, that is, it has a large number of methyl groups attached to DNA. The addition of methyl groups stops gene expression, so hypermethylated regions of DNA tend to express fewer genes. Facio-scapulo-humeral muscular dystrophy develops when the D4Z4 region has fewer attached methyl groups. In FSHD1, hypomethylation occurs because the D4Z4 region on one of the copies of chromosome 4 is abnormally short, with only 1 to 10 repeats instead of the usual 11 to 150 repeats. In FSHD2, hypomethylation is more frequently due to mutations in the SMCHD1 gene, located on the short arm of chromosome 18 (18p11.32), which encodes a protein involved in the regulation of gene activity by altering the structure of the DNA. Specifically, this protein plays a role in hypermethylation of the D4Z4 region near the end of chromosome 4. However, approximately 20% of people with FSHD2 do not have an identified mutation in the SMCHD1 gene, and the cause of hypomethylation is unknown.
Hypermethylation of the D4Z4 region normally maintains an open reading frame (ORF) called DUX4, located on chromosome 4, silenced in most adult cells and tissues. The DUX4 ORF is found in the segment of the D4Z4 region closest to the end of chromosome 4. In people with facioscapulo-humeral muscular dystrophy, hypomethylation of the D4Z4 region prevents the DUX4 ORF from being silenced in cells and tissues in which it is generally not expressed. Although little is known about the function of the ORF DUX4 when it is active, it is believed that it influences the activity of other genes, particularly in muscle cells.
More than a dozen mutations in the SMCHD1 gene have been identified as responsible for type 2 facio-scapulo-humeral muscular dystrophy. Changes in the SMCHD1 gene seem to play a role in both types: FSHD1 and FSHD2. Genetics in the SMCHD1 gene cause most cases of FSHD2. These mutations reduce the amount of SMCHD1 protein available to add methyl groups to the D4Z4 region. The resulting hypomethylation of this region prevents the DUX4 ORF from being silenced in cells and tissues in which it is generally not expressed, such as in adult muscle cells.
ORF-DUX4 is located next to a regulatory region of DNA on chromosome 4 known as a pLAM sequence, which is necessary for the encoding of the DUX4 protein. Some copies of chromosome 4 have a functional pLAM sequence, while others do not. Copies of chromosome 4 with a functional pLAM sequence are described as 4qA or "permissive". This variant acts as a polyadenylation signal (PAS) to stabilize the DUX4 transcript in the muscle of FSHD patients. In the 4qA region, the polymorphic sequence ATTAAA is required to act as a polyadenylation signal (PAS), which stabilizes the transcript and leads to the synthesis of the toxic protein. Those without a functional pLAM sequence are described as 4qB or "non-permissive." Without a functional pLAM sequence, no DUX4 protein is encoded. Because there are two copies of chromosome 4 in every cell, individuals can have two "permissive" copies of chromosome 4, two "non-permissive" copies, or one of each. Facioscapulo-humeral muscular dystrophy can only occur in people who have at least one "permissive" copy of chromosome 4. If an affected individual has a shortened D4Z4 region or an SMCHD1 gene mutation, the disease is expressed only if a pLAM functional sequence is also present to allow the DUX4 protein encoding. Additionally, mutations in the SMCHD1 gene, which cause FSHD2, are believed to also increase the severity of the disease in people with FSHD1. It is likely that the combination of a shortened D4Z4 region and an SMCHD1 gene mutation causes the D4Z4 region to have fewer attached methyl groups, allowing the DUX4 ORF to be highly active. Although the DUX4 protein is believed to influence the activity of other genes, particularly in muscle cells, it is not known how the presence of this protein damages or destroys these cells, leading to progressive muscle weakness and atrophy.
FSHD cases have also been related to certain chromosome 4 haplotypes, located at the subtelomeric level at 4q. In this sense, the existence of long variations in the sequence (SSLP: Single Sequence Length Polymorphism) of about 3 kb in D4Z4 has been studied, as well as the differences in a region rich in single nucleotide polymorphisms (SNPs: Single Nucleotide Polymorphisms) adjacent to the proximal end of D4Z4.
The analyzes of the relationship between the findings obtained, regarding these different possibilities, have allowed to find that in many patients with FSHD there is a relationship with the 4qA161 haplotype, although this haplotype has also been found in the unaffected control population. This 4qA161 haplotype differs from the 10qA166 haplotype corresponding to 10q (chromosome 10), which represents 96% of the alleles of this chromosome, with which this possible source of confusion could be excluded with considerable probability. The identification of the haplotype 4qA161 has a nucleotide G (guanosine) instead of A (adenosine) in SNP3, and a nucleotide of A (adenosine) instead of a nucleotide of G in SNP6. All FSHD cases have been found to have at least one of these two alleles, while in controls, about half of them have only one nucleotide of A (adenosine) in SNP3, and only one nucleotide of G (guanosine) in SNP6, so the 4qA161 allele would not exist. The allele 4qA161 has been found in 70% of patients with FSHD and in 33% of control cases, not affected by FSHD, so its presence indicates a considerably higher probability in favor of the diagnosis of FSHD.
Facioscapulo-humeral muscular dystrophy type 1 (FSHD1) is inherited in an autosomal dominant pattern, meaning that one copy of the shortened D4Z4 region on a "permissive" chromosome 4 is sufficient to express the disease. In most cases, an affected person inherits the altered chromosome from an affected parent. Other people with FSHD1 do not have a family history of the disease. These cases are described as sporadic and are due to a new D4Z4 contraction in a copy of a "permissive" chromosome 4. FSHD2 is inherited in a digenic pattern, which means that two independent genetic changes are required to express the disease. To have FSHD2, a person must inherit a mutation in the SMCHD1 gene and, separately, they must inherit one copy of a "permissive" chromosome 4. Affected individuals normally inherit the SMCHD1 gene mutation from one parent and the "permissive" chromosome 4 from the other parent.
Tests performed in IVAMI: At IVAMI for the diagnosis of FSHD type 1, we detected the 4qA161 haplotype, with its corresponding single nucleotide polymorphisms (SNPs), by means of PCR amplification and direct sequencing. Furthermore, the PAS (Polyadenylation signal) polymorphism in the pLAM region is studied by PCR and sequencing. The results of these two tests, when they are in accordance with those found in the cases of FSHD. In addition, for the diagnosis of FSHD type 2, we detect mutations in the SMCHD1 gene, through the complete amplification of all its exons and their subsequent sequencing.
Recommended samples: non-coagulated blood obtained with EDTA for separation of blood leucocytes, or a card with a dried blood sample (IVAMI can mail the card to deposit the blood sample).