boxesdux4 exons 1, 2, 3 arrows cornerpromotersstraightrna transcripts blacksenseredantisense bluedbe-tdashesdicing sites dux4 resides within the d4z4 macrosatellite repeat array, a series of tandemly repeated dna segments in the subtelomeric region (4q35) of chromosome 4. each d4z4 repeat is 3.3 kilobase pairs (kb) long and is the site of epigenetic regulation, containing both heterochromatin and euchromatin structures. in fshd, the heterochromatin structure is lost, becoming euchromatin. the subtelomeric region of chromosome 10q contains a tandem repeat structure highly homologous (99% identical) to 4q35, containing "d4z4-like" repeats with protein-coding regions identical to dux4 (d10z10 repeats and dux4l10, respectively). because 10q usually lacks a polyadenylation sequence, it is usually not implicated in disease. however, chromosomal rearrangements can occur between 4q and 10q repeat arrays, and involvement in disease is possible if a 4q d4z4 repeat and polyadenylation signal are transferred onto 10q, or if rearrangement causes fshd1. dux4 consists of three exons. exons 1 and 2 are in each repeat. exon 3 is in the plam region telomeric to the last partial repeat. multiple rna transcripts are produced from the d4z4 repeat array, both sense and antisense. some transcripts might be degraded in areas to produce si-like small rnas. some transcripts that originate centromeric to the d4z4 repeat array at the non-deleted element (nde), termed d4z4 regulatory element transcripts (dbe-t), could play a role in dux4 derepression. one proposed mechanism is that dbe-t leads to the recruitment of the trithorax-group protein ash1l, an increase in h3k36me2-methylation, and ultimately de-repression of 4q35 genes. fshd1 fshd involving deletion of d4z4 repeats (termed 'd4z4 contraction') on 4q is classified as fshd1, which accounts for 95% of fshd cases. typically, chromosome 4 includes between 11 and 150 d4z4 repeats. in fshd1, there are 1–10 d4z4 repeats. the number of repeats is roughly inversely related to disease severity. namely, those with 8 - 10 repeats tend to have the mildest presentations, sometimes with no symptoms; those with 4 - 7 repeats have moderate disease that is highly variable; and those with 1 - 3 repeats are more likely to have severe, atypical, and early-onset disease. deletion of the entire d4z4 repeat array does not result in fshd because then there are no complete copies of dux4 to be expressed, although other birth defects result. one contracted d4z4 repeat array with an adjoining 4qa allele is sufficient to cause disease, so inheritance is autosomal dominant. de novo (new) mutations are implicated in 10 - 30% of cases, 50% of which exhibit somatic mosaicism. it has been proposed that fshd1 undergoes anticipation, a trend primarily associated with trinucleotide repeat disorders in which disease manifestation worsens with each subsequent generation. as of 2019, more detailed studies are needed to definitively show whether or not anticipation plays a role. if anticipation does occur in fshd, the mechanism is different than that of trinucleotide repeat disorders, since d4z4 repeats are much larger than trinucleotide repeats, an underabundance of repeats (rather than overabundance) causes disease, and the repeat array size in fshd is stable across generations. d4z4 array examples, with each d4z4 repeat represented by a triangle. the circles above the triangles represent dna methylation, which determine dna packaging as represented by the circles in line with the triangles. fshd2 fshd without d4z4 contraction is classified as fshd2, which constitutes 5% of fshd cases. various mutations cause fshd2, all of chromatin modifier genes that result in d4z4 hypomethylation, at which the genetic mechanism converges with fshd1. approximately 80% of fshd2 cases are due to deactivating mutations in the gene smchd1 (structural maintenance of chromosomes flexible hinge domain containing 1) on chromosome 18. smchd1 is responsible for dna methylation, and its deactivation results in hypomethylation of the d4z4 repeat array. another cause of fshd2 is mutation in dnmt3b (dna methyltransferase 3b), which also plays a role in dna methylation. as of 2020, early evidence indicates that a third cause of fshd2 is mutation in both copies of the lrif1 gene, which encodes the protein ligand-dependent nuclear receptor-interacting factor 1 (lrif1). lrif1 is known to interact with the smchd1 protein. as of 2019, there are presumably additional mutations at other unidentified genetic locations that can cause fshd2. mutation of a single allele of smchd1 or dnmt3b can cause disease. mutation of both copies lrif1 has been tentatively shown to cause disease in a single person as of 2020. as in fshd1, a 4qa allele must be present for disease to result. however, unlike the d4z4 array, the genes implicated in fshd2 are not in proximity with the 4qa allele, and so they are inherited independently from the 4qa allele, resulting in a digenic inheritance pattern. for example, one parent without fshd can pass on an smchd1 mutation, and the other parent, also without fshd, can pass on a 4qa allele, bearing a child with fshd2. two ends of a disease spectrum initially, fshd1 and fshd2 were described as two separate genetic causes of the same disease. however, they can also be viewed not as distinct causes, but rather as risk factors. not rarely do both contribute to disease in the same individual. in those with fshd2, although they have do not have a 4qa allele with d4z4 repeat number less than 11, they still often have one less than 17 (relatively short compared to the general population), suggesting that a large number of d4z4 repeats can prevent the effects of an smchd1 mutation. further studies need to be done to determine the upper limit of d4z4 repeats in which fshd2 can occur. in those with a 4qa allele and 10 or fewer repeats, an additional smchd1 mutation has shown to worsen disease, classifying them as both fshd1 and fshd2. in these fshd1/fshd2 individuals, the methylation pattern of the d4z4 repeat array resembles that seen in fshd2. this combined fshd1/fshd2 presentation is most common in those with 9 - 10 repeats, and is seldom found in those with 8 or less repeats. the relative abundance of smchd1 mutations in the 9 - 10 repeat group is likely because a sizable portion of the general population has 9 - 10 repeats with no disease, yet with the additive effect of an smchd1 mutation, symptoms develop and a diagnosis is made. in those with 8 or fewer repeats, symptoms are more likely than in those with 9 - 10 repeats, leading to diagnosis regardless of an additional smchd1 mutation. the apparent frequency of fshd1/fshd2 cases in the 9 - 10 repeat range, combined with the fshd2-like methylation pattern, suggest the 9 - 10 repeat size to be an overlap zone between fshd1 and fsdh2. pathophysiology molecular dux4 signaling in fshd-affected skeletal muscle. as of 2020, there seems to be a consensus that aberrant expression of dux4 in muscle is the cause of fshd. dux4 is expressed in extremely small amounts, detectable in 1 out of every 1000 immature muscle cells (myoblast), which appears to increase after myoblast maturation, in part because the cells fuse as they mature, and a single nucleus expressing dux4 can provide dux4 protein to neighboring nuclei from fused cells. it remains an area of active research how dux4 protein causes muscle damage. dux4 protein is a transcription factor that regulates many other genes. some of these genes are involved in apoptosis, such as p53, p21, myc, and β-catenin, and indeed it seems that dux4 protein makes muscle cells more prone to apoptosis. other dux4 protein-regulated genes are involved in oxidative stress, and indeed it seems that dux4 expression lowers muscle cell tolerance of oxidative stress. variation in the ability of individual muscles to handle oxidative stress could partially explain the muscle involvement patterns of fshd. dux4 protein downregulates many genes involved in muscle development, including myod, myogenin, desmin, and pax7, and indeed dux4 expression has shown to reduce muscle cell proliferation, differentiation, and fusion. dux4 protein regulates a few genes that are involved in rna quality control, and indeed dux4 expression has been shown to cause accumulation of rna with subsequent apoptosis. estrogen seems to play a role in modifying dux4 protein effects on muscle differentiation, which could explain why females are lesser affected than males. the cellular hypoxia response has been reported in a single study to be the main driver of dux4 protein-induced muscle cell death. the hypoxia-inducible factors (hifs) are upregulated by dux4 protein, possibly causing pathologic signaling leading to cell death. another study found that dux4 expression in muscle cells led to the recruitment and alteration of fibrous/fat progenitor cells, which helps explain why muscles become replaced by fat and fibrous tissue. a single study implicated ripk3 in dux4-mediated cell death. muscle histology microscopic cross-sectional views of fshd-affected muscle fibers. visible is inflammation and fibrosis, as well as muscle fiber shape change, death, and regeneration. unlike other muscular dystrophies, early muscle biopsies show only mild degrees of fibrosis, muscle fiber hypertrophy, and displacement of nuclei from myofiber peripheries (central nucleation). more often found is inflammation. there can be endomysial inflammation, primarily composed of cd8+ t-cells, although these cells do not seem to directly cause muscle fiber death. endomysial blood vessels can be surrounded by inflammation, which is relatively unique to fshd, and this inflammation contains cd4+ t-cells. inflammation is succeeded by deposition of fat (fatty infiltration), then fibrosis. individual muscle fibers can appear whorled, moth-eaten, and, especially, lobulated. muscle involvement pattern why certain muscles are preferentially affected in fshd remains unknown. there are multiple trends of involvement seen in fshd, possibly hinting at underlying pathophysiology. individual muscles can weaken while adjacent muscles remain healthy. the right shoulder and arm muscles are more often affected than the left upper extremity muscles, a pattern also seen in poland syndrome and hereditary neuralgic amyotrophy; this could reflect a genetic, developmental/anatomic, or functional-related mechanism. the deltoid is often spared, which is not seen in any other condition that affects the muscles around the scapula. medical imaging (ct and mri) have shown muscle involvement not readily apparent otherwise a single mri study shows the teres major muscle to be commonly affected. the semimembranosus muscle, part of the hamstrings, is commonly affected, deemed by one author to be "the most frequently and severely affected muscle." of the quadriceps muscles, the rectus femoris is preferentially affected of the gastrocnemius, the medial section is preferentially affected; the iliopsoas, a hip flexor muscle, is very often spared. retinopathy tortuosity of the retinal arterioles, and less often microaneurysms and telangiectasia, are commonly found in fshd. abnormalities of the capillaries and venules are not observed. one theory for why the arterioles are selectively affected is that they contain smooth muscle. the degree of d4z4 contraction correlates to the severity of tortuosity of arterioles. it has been hypothesized that retinopathy is due to dux4-protein-induced modulation of the cxcr4–sdf1 axis, which has a role in endothelial tip cell morphology and vascular branching. diagnosis american academy of neurology (ann) guidelines for genetic testing for suspected fshd. not all laboratories follow this workflow.fshd can be presumptively diagnosed in many cases based on signs, symptoms, and/or non-genetic medical tests. genetic testing can provide definitive diagnosis. in the absence of an established family history of fshd, diagnosis can be difficult due to the variability in how fshd manifests. genetic testing genetic testing is the gold standard for fshd diagnosis, as it is the most sensitive and specific test available. commonly, fshd1 is tested for first. a shortened d4z4 array length (ecori length of 10 kb to 38 kb) with an adjacent 4qa allele supports fshd1. if fshd1 is not present, commonly fshd2 is tested for next by assessing methylation at 4q35. low methylation (less than 20%) in the context of a 4qa allele is sufficient for diagnosis. the specific mutation, usually one of various smchd1 mutations, can be identified with next-generation sequencing (ngs). assessing d4z4 length measuring d4z4 length is technically challenging due to the d4z4 repeat array consisting of long, repetitive elements. for example, ngs is not useful for assessing d4z4 length, because it breaks dna into fragments before reading them, and it is unclear from which d4z4 repeat each sequenced fragment came. in 2020, optical mapping became available for measuring d4z4 array length, which is more precise and less labor-intensive than southern blot. molecular combing is also available for assessing d4z4 array length. these methods, which physical measure the size of the d4z4 repeat