Detection of genes causing Fibromyalgia Sierra Hamlet

What is fibromyalgia syndrome (FMS)? A chronic pain disorder Characterized by widespread musculoskeletal pain, fatigue, and tenderness in localized areas Affects 2-5% of the general population, mostly females Affects a wide range of ages, but generally affects people over the age of 30 Underlying cause is not completely understood Evidence suggests that it has a familial trend

Fibromyalgia Tender Points Fibromyalgia is difficult to diagnose because pain is highly personal. One person’s ”pain all over” isn’t necessarily the same as another person’s “pain all over.” The American College of Rheumatology guidelines suggest that people with fibromyalgia have pain in at least 11 of 18 localized tender points, or they may spread pain to other parts of the body Doctor applies a certain amount of pressure to each tender point in the exam

Fibromyalgia Family Study 116 multicase families with fibromyalgia were recruited If the primary patient is female (male), the first-degree relative of the patient must be a parent or offspring and an unaffected female (male) sibling of the patient must be available Only subjects who were 12 years or older were included

Sibling recurrence risk The proportion of affected siblings among all siblings of affected patients with fibromyalgia Used to determine whether or not there was an excess risk among family members Assumed a population prevalence of 2% The sibling recurrence risk was adjusted for sampling bias because families were recruited via an affected individual and the size of sibships varies from family to family 342 siblings were included in assessment, 194 of whom were female Sibling recurrence risk of 27.2%, ratio of 13.6, was determined for whole set of 342 The risk was higher for only the 194 females (43.8%, ratio of 12.9) Suggests a strong genetic component of fibromyalgia Not all of the families are the same size. In fact, one family had 13 siblings involved, which was the largest. Was first estimated then corrected with an equation including the number of families, number of offspring, and the number of patients affected Because the prevalence of fibromyalgia is higher in women than in men in the general population, the corresponding sibling recurrecnce risk ration for only the females was 12.9. Overall, these results suggest a strong genetic component of fibromyalgia.

Genome-wide linkage scan Genotyped 341 markers on 22 autosomal chromosomes in 203 affected sibling pairs One chromosome region on chromosome 17 (17p11.2-q11.2) showed a nominal P value less than 0.00074, which is the criterion for genome-wide suggestive linkage This region coincides with the map coordinate for two potential candidate genes for fibromyalgia, SLC6A4 and TRPV2 The chromosome 17p11.2-17q11.2 region coincides with the map coordinate for two potential candidate genes for fibromyalgia: the serotonin transporter gene (SLC6A4) and the transient receptor potential vanilloid channel 2 gene (TRPV2)

Serotonin transporter gene (SLC6A4) SLC6A4 encodes the human serotonin transporter protein and is mapped to chromosome 17q11.1-q12.12 A functional polymorphism in the 5’ regulatory region of SLC6A4 involves 2 major alleles, S (short) and L (long), that correspond to the presence of 14 or 16 repeat units of a 20-23 bp incomplete repeat The short allele was observed to reduce transcription efficiency for SLC6A4, resulting in decreased gene expression and decreased serotonin uptake in lymphoblast cell lines Further studies are needed to relate the role of SLC6A4 variants to fibromyalgia Association studies of this functional polymorphism and clinical pain syndroms have generated conflicting results so further studies are needed

Transient receptor potential vanilloid channel 2 gene (TRPV2) Data supports the contribution of many members of the TRPV family to pain, including TRPV-1, TRPV-4, and TRPA-1 Specifically pathologic pain associated with inflammatory and neuropathic states TRPV2 may play a role in mediating pain, but more investigation must be done to understand the role of the gene in pain biology and chronic pain disorders like fibromyalgia

Discovery of Potential New Gene Variants and Inflammatory Cytokine Associations with FMS It is possible that the widespread pain observed in FMS is caused by an underlying factor such as chronic inflammation rather than only defects in pain-related genes 150 unrelated patients with fibromyalgia and their parents were recruited The ages and characteristics of the patients varied Musculoskeletal pains had existed in the patients for over three months and were accompanied by complaints from one or more other systems including the central nervous system, gastrointestinal system, and the genitourinary tract At least 11 of the 18 predetermined pressure points were elicited in each patient

MEFV gene Became interested in the MEFV gene while searching for possible genes that relate to fibromyalgia on an inflammatory basis Mutations in the MEFV gene cause Familial Mediterranean Fever (FMF) FMS and FMF share overlapping symptoms, including unexplained abdominal pain in FMF and high prevalence of irritable bowel syndrome in FMS, both of which suggest an inflammatory condition Sequenced exons and splice junctions as regions of likely functional significance in MEFV in 100 fibromyalgia patients and their parents Discovered that rare missense variants of the MEFV gene are collectively associated with risk of FMS as they are present in 15% of FMS patients Findings supported their hypothesis regarding a genetic link to an inflammatory basis for the syndrome

Exome Sequencing Genomic DNA from 19 FMS patients was extracted from peripheral blood lymphocytes A total of 790,000 SNPs were observed, average of 82,738 per sample 66,902 were missense mutations, nonsense mutations, or affecting splicing sites A filtering strategy was applied to the large number of SNPs Only variants producing missense, nonsense, or splice variants (66,902) Only variants found in at least 2 of the 19 patients (18,000) Only SNPs found in the May 2011 release of the 1000 Genomes database (12,000) Variants with a population frequency of less than or equal to 2.5% (905) Variants likely to produce the most severe effect on phenotype, specifically nonsense mutations (7)

Transmission Disequilibrium Test (TDT) Measures the over-transmission of an allele from heterozygous parents to affected offspring Parents serve as “controls,” in that transmission of heterozygous rare alleles from parent to offspring should occur at a probability of >0.5 to be associated with FMS TDT was applied to the 7 genes that remained after the filtering strategy previously described Direct sequence analysis of each gene in the 300 parents revealed that 13-33 of the parents were heterozygous for each gene

Mutation Analysis Direct sequence analysis of the 150 patients plus their parents showed complete concordance with the exome sequencing data of the original 19 patients TDT was then applied to all 450 samples for each of the 7 genes C11orf40 and ZNF77 showed as significantly associated genes

Chromosome 11 Open Reading Frame 40 (C11orf40) The nonsense mutation, W32X, was identified in the C11orf40 gene in 2 out of 19 FMS patients by exome sequencing Directed sequencing of the other 131 patients showed 17 additional heterozygous patients and 1 homozygous patient to the C11orf40 nonsense mutation A total of 20 out of 150 patients and 27 out of 300 parents carried the W32X variant Transmission analysis of the W32X variant shows that the mutant allele was transmitted from parent to affected offspring in 19 of these 27 events, at a probability of 0.70 Thus, the mutant allele encoding the W32X mutation for the C11orf40 gene was associated with FMS C11orf40 is expressed in normal human brain, heart, lung and ovary tissues, but its function is unknown

Zinc Finger Protein 77 (ZNF77) 17 out of the 150 FMS patients were heterozygous for the nonsense mutation, Q100X Direct sequence analysis revealed 24 heterozygous parents that carried the nonsense mutation TDT analysis then revealed that the mutant allele was transmitted to the affected offspring in 17 of 24 events, also giving a ratio of 0.70 Thus, the nonsense mutant allele of the ZNF77 gene was also associated with FMS Analysis of the gene expression of ZNF77 shows that it was expressed in all normal human tissues, but the functions of most of the ZNF genes are unknown

References Arnold, Lesley M., et al. “The Fibromyalgia Family Study: A Genome-Scan Linkage Study.” Arthritis and Rheumatism, U.S. National Library of Medicine, Apr. 2013, www.ncbi.nlm.nih.gov/pmc/articles/PMC3618544/. Driver, MD Catherine Burt. (2017) “Fibromyalgia Causes, Symptoms, Treatment & Diagnosis.” MedicineNet, https://www.medicinenet.com/fibromyalgia_facts/article.htm#what_i s_the_treatment_for_fibromyalgia. Feng J, Zhang Z, Wu X, Mao A, Chang F, Deng X, et al. (2013) Discovery of Potential New Gene Variants and Inflammatory Cytokine Associations with Fibromyalgia Syndrome by Whole Exome Sequencing. PLoS ONE 8(6): e65033. https://doi.org/10.1371/journal.pone.0065033