Facioscapulohumeral Muscular Dystrophy (FSHD) Molecular Diagnosis: from Traditional Technology to the NGS Era

neurogenetics (2019) 20:57–64 https://doi.org/10.1007/s10048-019-00575-4

REVIEW ARTICLE

Facioscapulohumeral muscular dystrophy (FSHD) molecular diagnosis: from traditional technology to the NGS era

Stefania Zampatti1 & Luca Colantoni1 & Claudia Strafella2 & Rosaria Maria Galota1 & Valerio Caputo2 & Giulia Campoli1 & Giulia Pagliaroli1 & Stefania Carboni1 & Julia Mela1 & Cristina Peconi1 & Stefano Gambardella3 & Raffaella Cascella1,4 & Emiliano Giardina1,2

Received: 14 January 2019 /Accepted: 17 March 2019 /Published online: 25 March 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract Facioscapulohumeral muscular dystrophy (FSHD) is a genetic neuromuscular disorder which mainly affects the muscles of the face, shoulder, and upper arms. FSHD is generally associated with the contraction of D4Z4 macrosatellite repeats on 4q35 chromosome or mutations in SMCHD1, which are responsible of the toxic expression of DUX4 in muscle tissue. Despite the recent application of NGS techniques in the clinical practice, the molecular diagnosis of FSHD is still performed with dated techniques such as Southern blotting. The diagnosis of FSHD requires therefore specific skills on both modern and less modern analytical protocols. Considering that clinical and molecular diagnosis of FSHD is challenging, it is not surprising that only few laboratories offer a comprehensive characterization of FSHD, which requires the education of professionals on traditional techniques even in the era of NGS. In conclusion, the study of FSHD provides an excellent example of using classical and modern molecular technologies which are equally necessary for the analysis of DNA repetitive traits associated with specific disorders.

Keywords FSHD . D4Z4 contraction . SMCHD1 . Genetic counseling . Genetic test

Introduction array of tandemly repeated units known as D4Z4. However, approximately 5% of patients display the typical disease phe- Facioscapulohumeral muscular dystrophy (FSHD, OMIM no. notype without carrying the D4Z4 contraction. This alterna- 158900) is the third most common hereditary myopathy, after tive form is termed FSHD2 and it has been associated with Duchenne dystrophy and myotonic dystrophy [1]. It has been pathogenetic mutations in Structural Maintenance of estimated to affect 1 in 8333 people worldwide [2]. Linkage Chromosomes Flexible Hinge Domain-Containing 1 analysis allowed the identification of the FSHD genetic locus (SMCHD1, 18p11.32) and DNA Methyltransferase 3 Beta on 4q35 chromosome [3]. The classical form of the disease (DNMT3B, 20q11.21) genes [4]. Although two distinct forms (referred to as FSHD1), in fact, has been associated with DNA of the disease (FSHD1 and FSHD2) have been described, rearrangements (contraction) in this region, consisting of an recent literature indicated that FSHD2-associated genes can also act as disease modifiers in FSHD1, rising the need for testing FSHD2 genes even in FSHD1 patients [3, 4]. * Raffaella Cascella The molecular diagnosis of FSHD1 is still based on tradi- [email protected] tional methods as Southern blotting that is expensive and labor intensive and requires large amounts of DNA. Several efforts 1 Molecular Genetics Laboratory UILDM, Santa Lucia Foundation, have been made to set up alternative molecular methods, such via Ardeatina 354, 00142 Rome, Italy as molecular combing (MC) [5]. On the other hand, charac- 2 Department of Biomedicine and Prevention, Tor Vergata University, terization of FSHD2-related genes is made either by tradition- via Montpellier 1, 00133 Rome, Italy al or modern technologies (direct sequencing and NGS, re- 3 IRCCS Neuromed, via Atinense, 18, 86077 Pozzilli, Italy spectively) which allow high-throughput, less expensive, 4 Department of Biomedical Sciences, Catholic University Our Lady and reliable results. In this context, prominent attention should of Good Counsel, Rruga Dritan Hoxha, 1000 Tirana, Albania be given to the training of biologists and laboratory 58 Neurogenetics (2019) 20:57–64 technicians which should properly know not only the latest Although some severity markers have been recently de- but also the oldest technologies available to perform molecu- scribed, in most of the cases, the prognosis of the disease is lar diagnosis of peculiar disorders. unpredictable [10]. Sudden and dramatic worsening of the disease can occur after long periods of stable conditions. In addition, the clinical spectrum is widely variable among unre- Clinical features lated patients, including subjects with slight muscle weakness, who are unaware of being affected, as well as patients who are FSHD is a slowly progressive muscular dystrophy that typi- wheelchair-dependent [8]. This variability in disease clinical cally involves facial and shoulder muscles. The clinical course expression has been exemplified by a study on monozygotic of the disease can be highly variable, alternating stable and male twins, who carry the same genetic mutation but are af- worse conditions. The transmission pattern generally follows fected by FSHD at dramatically different extents [4]. Before an autosomal dominant pattern, although reduced penetrance the advent of molecular diagnosis, penetrance of FSHD was and variable expressivity complicate the identification of af- evaluated considering the clinical examination of patients at fected subjects within families [1]. In addition, up to 30% of risk and their age, resulting to be between 83 and 95% at cases are due to de novo mutations and, approximately half of 20 years of age [11]. However, a higher penetrance of the them, result from a post-zygotic mutation leading to mosai- disease has been reported in males compared to females, cism [4, 6]. The onset is usually between 6 and 20 years, who appeared to be mostly asymptomatic or minimally affect- suddenly in the adulthood. In the early stage of the disease, ed [7]. Recently, a prospective cross-sectional study reported the muscle involvement can be asymmetrical, and the weak- the age of onset of FSHD as a severity marker. Patients with an ness is so slight and slowly progressive that many cases re- early-onset FSHD (observed in 7–15% of all FSHD cases) main subclinical for several years. Generally, the first signs of have been found to experience a more severe and progressive the disease include difficulties in raising the arms over the disease with 57% patients being dependent on wheelchair head and scapular winging. Facial weakness has also been compared to 20–30% of classical FSHD cases. In addition, observed at the initial clinical stage of the disease and, in some age at onset has also been described as a prognostic marker cases, many years before the diagnosis. The involvement of in relation to the frequency of systemic complications [10]. facial muscles is generally underestimated because of the little impact on activities of daily living. The affected facial muscles are the orbicularis oculi, the zygomaticus, and the orbicularis Genetic aspects of FSHD1 oris which appear weak and thin. Patients are therefore unable to firmly close the eyes, to inflate cheeks, and to whistle. FSHD1 has been associated with DNA rearrangements Masticatory, lingual, and extraocular muscles are not involved (contraction) in a polymorphic region known as D4Z4 in FSHD [4]. The scapular girdle is the most involved region: (4q35) that is characterized by an array of tandemly repeated trapezius and pectoral muscles at the initial stage; the units extending for 3.3 kb. In normal conditions, the D4Z4 sternocleidomastoid, serratus magnus, rhomboid, erector array varies from 10 to 100 repeated units (RU), whereas spinae, latissimus dorsi later on; and ultimately, the deltoid FSHD patients have less than 10 RU [12]. The number of muscles [7]. Shoulder bones protrude out on the back, causing repeats can be determined by Southern blotting and hybridi- thereby scapular winging and clavicles sticking out and zation with the p13E-11 probe, which is able to recognize the rounded shoulders. Biceps are usually less involved than tri- D4Z4 region. However, the interpretation of the p13E-11 hy- ceps and brachioradialis muscles, although biceps can present bridization pattern is complicated by the fact that the probe a thinner proximal trait compared to the distal one (BPopeye^ can also recognize an additional locus on the 10q26 region effect) [8]. Pelvic girdle is involved some years after the onset that is nearly completely homologous to the 4q35 region (> of the disease, leading to pelvic muscle weakness and, subse- 98%) but it is not related to FSHD1 [4]. Moreover, the high quently, to mild lordosis and pelvic instability. Pretibial in- degree of sequence homology between 4qter and 10qter loci volvement leads to foot drop and gait instability [8]. facilitates inter-chromosomal exchange resulting in total and Beevor’s sign (the abnormal upward movement of the umbi- partial translocations. These translocation alleles are derive licus when the patient raises the head from a supine position) from ancient founding events but, indeed, complicate even is typical in the advanced stage of the disease, when up to 20% more the molecular diagnosis of the disease [6, 13]. of patients become wheelchair-dependent [7]. Cardiac in- Many studies suggested that D4Z4 contractions are not the volvement (rhythm disorders and cardiomegaly) is rare. only required conditions for FSHD manifestation [14]. The Although some studies have hypothesized anticipation phe- genomic architecture of the 4q35 region and its flanking re- nomenon, no molecular and clinical findings confirmed this gions has been therefore deeply investigated, in order to search hypothesis and phenotype worsening in subsequent genera- for other variables contributing to FSHD etiopathogenesis. Two tions remains a rare event [9]. sub-telomeric variations distal to D4Z4 were identified on Neurogenetics (2019) 20:57–64 59 chromosome 4, namely 4qA and 4qB alleles. Both are equally potential complications. In this context, the D4Z4 deletion size distributed in the general population, but only 4qA has been appears to be somewhat predictive of the overall rate of dis- associated with FSHD and it is referred to as Bpermissive allele^ ease progression, although other sources of variation can af- [15]. fect disease severity and frequency of systemic features. Proximal to D4Z4 array, a simple sequence length poly- Usually, patients with severe early-onset disease have 1–3- morphism (SSLP) exists, whose sequence length can range RU repeats, suggesting a much more robust correlation be- between 157 and 180 bp. The analysis of 4q sub-telomeric tween disease severity and larger D4Z4 contractions [10]. variants flanking the D4Z4 array revealed different haplotypes on 4qter, including 4A159, 4A161, 4B163, 4A166, and 4A168. Currently, all 4qA alleles are pathogenic except for Genetic aspect of FSHD2 4A166. Case-control studies revealed a strong association between FSHD and 4qA161, which is the most common allele Up to 5% of FSHD patients do not show D4Z4 contraction on contributing to create a Bpermissive^ genetic background for 4q35. These patients show identical clinical features of disease etiopathogenesis [12, 15]. On this subject, 4A159, FSHD1 cases and carry similar DNA hypomethylation levels 4A163, 4A166H, and 4A168 haplotypes have also been de- and heterochromatin markers on D4Z4 repeats, but they differ scribed as uncommon permissive alleles [7]. for other genetic signatures [24]. This alternative form of the Moreover, each unit of the D4Z4 array contains a copy of disease is referred to as FSHD2 (OMIM no. 158901) and has the retrogene Double Homeobox 4 (DUX4,OMIMno. been recognized to be distinct and much less common than the 606009), which is normally expressed during early embryonic classical type. In FSHD2 patients, D4Z4 hypomethylation is development and in testis whereas it is silenced in somatic observed on all D4Z4 repeat arrays without D4Z4 contrac- tissue [15]. Somatic repression of DUX4 requires a combina- tions, although the repeat size is usually smaller or borderline tion of epigenetic mechanisms necessary for maintaining a (between 8 and 20 RU), making the region more susceptible to repressive chromatin condition. In FSHD1, instead, the further hypomethylation [25, 26]. More than 80% of FSHD2 contracted allele induces a chromatin relaxation which results cases are caused by the inheritance of two independent genetic in the hypomethylation of the D4Z4 and a variegated expres- variations: a heterozygous loss-of-function mutation in sion of DUX4 in myonuclei [16]. DUX4 expression has been SMCHD1 (OMIM no. 614982) gene and a 4qA permissive found to be toxic in muscle tissue, hampering myogenic dif- allele with the PAS inducing the toxic expression of DUX4 ferentiation, increasing oxidative stress and, ultimately, caus- [18, 27, 28]. ing muscle atrophy [17]. Interestingly, DUX4 transcripts are The encoded protein by SMCHD1 is a distant member of produced only in the presence of a polymorphic sequence the highly conserved SMC protein family that is an essential (ATTAAA) residing in the 4qA variant that acts as a component of the cohesin/condensin protein complexes [3]. polyadenylation signal (PAS) to stabilize the transcript of In muscle cells, SMCHD1 has been reported to directly bind DUX4 in muscle tissue of FSHD patients [18]. In contrast, the D4Z4 repeat array, suggesting that it is essential for the lack of PAS in 4qB or 10q alleles does not allow the maintaining a repressive chromatin structure in somatic aberrant expression of DUX4 in muscles, although 4qB is cells [18]. More than 52 SMCHD1 mutations associated known to permit the physiological expression of DUX4 in with FSHD have been reported up to date [28]. These mu- early embryonic development (at 4-cell stage) likewise 4qA tations decrease the DNA binding activity or the enzymatic alleles [4, 19–23]. The size of the 4q array is determined by activity of SMCHD1, resulting in hypomethylation and re- digestion with the restriction enzyme EcoRI, which generates laxation of chromatin in all D4Z4 repeat arrays on chromo- fragments of variable size in relation to the number of D4Z4 some 4. In the presence of a 4qA allele, this is thought to repeats. EcoRI fragments are considered as one the key ele- bring enhancers and promoters (which are normally kept ments to determine the clinical phenotype, age of onset, dis- away by SMCHD1) closer enough to interact and contribute ease course, and age at loss of ambulation [4, 7]. An inverse to create a transcriptionally permissive environment for the relationship exists between the D4Z4 repeat size and the se- expression of DUX4 [18]. However, SMCHD1 mutations do verity and progression of disease. In fact, individuals with ≥ not cause FSHD2 when combined with non-permissive 4qB 11 RU (> 43 kb) are normally unaffected, whereas 1–3-RU alleles, suggesting the existence of a digenic mechanism for (10–17 kb) fragments are associated with more severe FSHD1 FSHD2 pathogenesis. Approximately 20% of FSHD2 indi- [1]. Difficulties in predicting the disease severity have been viduals carrying hypomethylation at D4Z4,aSMCHD1 mu- encountered in patients carrying 4–7-RU (20–30 kb) frag- tation, and a permissive 4q haplotype are asymptomatic, ments because of the high clinical heterogeneity, although this indicating thereby the existence of incomplete penetrance repeat size is frequently observed among FSHD1 patients. as for FSHD1 [28]. Some FSHD families have been identi- The availability of predicting factors for disease severity is fied with both FSHD1- and FSHD2-associated mutations. critical for genetic counseling, treatment, or prevention of These patients appear to be more severely affected with 60 Neurogenetics (2019) 20:57–64 respect to individuals carrying only the D4Z4 contraction. early stages of FSHD can be promptly treated with some This observation showed that SMCHD1 can act as a disease physiotherapy interventions aimed to prevent the acquisi- modifier in FSHD1 patients [29]. In addition to SMCHD1, tion of harmful habits. However, patients sometimes pre- other environmental and/or (epi)genetic factors are likely to fer to avoid genetic testing, because of the lack of well- be involved in modifying disease severity and clinical man- defined therapies. The details about technologies are al- ifestation. On this subject, exome sequencing was per- ways difficult to explain to patients, because of the labo- formed on eight FSHD families carrying D4Z4 hypomethy- rious molecular approach utilized for FSHD diagnosis. In lation without evidence of a pathogenic SMCHD1 mutation. particular, the counselor should explain all of the analyt- In two families, a potentially damaging heterozygous vari- ical steps performed to achieve reliable results, including ant in DNMT3B (OMIM no. 602900) gene was identified both traditional (Southern blotting and PFGE (pulsed-field [30].ThegeneencodesaknownD4Z4-chromatin modifier, gel electrophoresis); LGE (linear gel electrophoresis)) and suggesting that DNMT3B mutations may act as disease next-generation sequencing (NGS) methods. Furthermore, modifiers in FSHD patients. As for SMCHD1, the effect of the identification of D4Z4-deleted alleles on the repetitive DNMT3B mutations on DUX4 expression and disease man- region of 4q35 should be confirmed with a segregation ifestation strictly depends on the presence of PAS and on the analysis (if available), especially when the identification size of the D4Z4 repeat array [4]. of a short allele does not correlate with clinical features because of incomplete penetrance. In this case, a detailed pre-test genetic counseling should be offered to each fam- Genetic counseling in FSHD ily member that undergoes genetic test, paying attention to explain the possible occurrence of this event. Clinical and molecular characteristics of FSHD have to be Moreover, segregation analysis can have other implica- considered in order to provide patients and their families with tions since familial links might not be confirmed (lack an adequate genetic counseling. Considering that FSHD is an of biological paternity). If this information is not impor- autosomal dominant disorder with variable penetrance and tant for the explanation of the disorder within the family, expressivity, efforts to find measurable factors able to discrim- counselors may decide to not communicate it, unless ex- inate FSHD patients have been so far tried with limited suc- plicitly requested by the patient before undertaking the cess. In particular, some specific genotypes (such as the test. The time required for molecular characterization is shortest D4Z4 allele) have been associated with a more severe hard to predict. In a family in which the disorder co- phenotype and a higher disease penetrance [31–34]. On the segregates with a contracted 4q35 allele, the test to be other hand, longer alleles seem to be less penetrant. In the performed in family members is quite easy and exclusive- clinical practice, the lack of a precise genotype-phenotype ly based on the detection of the known molecular defect. association and the high intra-familial variability make the Sporadic cases, which are negative to D4Z4 contraction, genetic counseling very difficult [14]. are usually screened for mutations in SMCHD1 and The genetic counseling should be offered to each pa- DNMT3B, requiring thereby longer times for analysis. tient with a suspicion or a clinical diagnosis of FSHD. Furthermore, epigenetic evaluations could be useful in Pre-test and post-test genetic counseling should be per- selected cases, to identify the cause of FSHD. formed in each case. Patients should be adequately in- Occasionally, the first-level test (Southern blot and formed concerning the available molecular tests, the pos- PFGE, LGE) and the segregation analyses could reveal a sible expected and unexpected findings, and their impli- non-canonical segregation pattern due to translocation cations for their own health and of their relatives, in order events, hampering thereby the achievement of a conclu- to enable them to make an informed choice when consid- sive diagnosis, because of the lack of molecular tech- ering molecular testing [35, 36]. Given FSHD penetrance niques able to differentiate between different rearrange- and expressivity, it is recommended that patients fully ments [39, 40]. During the pre-test genetic counseling, understand the main implications of a positive result be- the counselor should also provide an anticipation of the fore undertaking the test. During pre-test genetic counsel- final report including the results of the genetic test, in ing, it is essential to address the following topics: (i) the order to get patients ready for the interpretation of the test is optional but can support or confirm the clinical reported data. The post-test genetic counseling is focused diagnosis, (ii) the technologies which are applied, (iii) on the explanation of the final report and the clinical limitations (sensitivity and specificity) of the test, (iv) significance of the results for patients and their family incidental findings, (v) the timing, (vi) presentation of members. Patients should be informed about the risk of the final report [37, 38]. During the diagnostic procedure, transmission of pathogenic mutations and the recurrence the molecular confirmation of FSHD can be helpful, es- risk of disease in case of identification of the disease- pecially to make an early diagnosis of disease. In fact, associated mutation. Penetrance and variable expressivity Neurogenetics (2019) 20:57–64 61 should be extensively discussed during post-test genetic the DNA fragments from 4q35 and 10q26, and quantify counseling. Although an accurate genotype-phenotype post-zygotic mosaicisms in FSHD patients. However, this ap- prevision is not possible, patients can make prenatal test- proach needs to be further validated and refined before to be ing to detect the pathogenic mutation on the DNA of the implemented into the clinical practice [42, 43]. fetus. In this case, the counselor should inform the patient Currently, no effective therapies are available to prevent about the limitations of the genetic test. In fact, a prenatal FSHD. In this perspective, the usefulness of pre- test for FSHD can only search for a disease-associated symptomatic genetic test should be discussed during genetic mutation, but it cannot predict the phenotype of the fetus, counseling [44]. On this subject, it is usually recommended to because of incomplete penetrance and variable expressiv- perform a neurological evaluation before genetic testing. ity of FSHD. Sometimes, a psychological evaluation of patients and/or their Pre-implantation genetic diagnosis (PGD) is theoretically familial members can be performed before the genetic test, in possible, but the big amount of DNA required for the PFGE order to avoid harmful implications of the possible results. analysis is unsuited to the technologies utilized for PGD. This Further studies are needed to detect individual trajectories of is the reason why PGD may be performed through linkage disease and response to therapies/environmental factors as analyses, but the detection of short D4Z4 alleles at 4q35 well as to identify genetic signatures which can be utilized (FSHD1) is generally not feasible due to the chromosomal for predictive purposes. Moreover, the lack of a genomic per- position of the repetitive trait. Limitations of the linkage anal- sonalized medicine in FSHD often influences testing and ysis for detecting 4q alleles should be communicated. In fact, medical decisions. the D4Z4 region is located at the telomeric end of the long arm of chromosome 4, making the selection of appropriate short tandem repeat (STR) markers to perform linkage analysis dif- Conclusions ficult. In addition, STRs are very rare within the region of interest, complicating the possibility of investigating other In the last 10 years, the availability of new-generation tech- markers in case of non-informative results. Furthermore, the nologies has been crucial for an extensive and deep investiga- great genomic distance between the D4Z4 region and STRs tion of the human genome in health and disease conditions. A significantly increases the recombination risk that should al- new era of genetics therefore originated, moving from stem ways be considered when PGD or prenatal diagnosis is per- cells to gene therapy, and gene editing is becoming really formed. Invasive prenatal testing is therefore recommended in promising. In this context, genetics progress was very useful each PGD for FSHD, in order to verify the occurrence of to understand the pathogenic pathways of disease and to de- recombination events [41]. Concerning the analysis of velop innovative genome-based tools to provide patients with SMCHD1 for PGD, it is currently not recommended into the more effective and safer treatments [45]. clinical practice. However, it may be useful in families show- In 1992, the cause of FSHD was shown to be the deletion ing a peculiar segregation pattern associated with a known of a region within chromosome 4 that consists of repeated mutation in SMCHD1. Recently, new molecular technologies DNA called D4Z4. At that time, many scientists assumed that showed promising results in the detection of FSHD1 molecu- FSHD originates by a mechanism observed for other genetic lar signature. MC and optical mapping approaches seem to be disorders: the mutation of a gene within D4Z4 makes it unable the most feasible alternative methods for FSHD molecular to produce the corresponding protein. However, subsequent diagnosis. In particular, MC allows the direct visualization research proved exactly the opposite: FSHD is not due to the and cartography of numerous individual DNA molecules at loss of a protein, but to its excessive production. The subse- 1-kb resolution. Although MC was originally developed to quent step was to understand how D4Z4 was able to regulate map genes for positional cloning and to study DNA replica- protein production in FSHD. tion, it could be helpful for the diagnosis of disease involving Also, the model of inheritance of FSHD seems to fol- complex chromosomal rearrangements, including FSHD. In low the classical Mendelian patterns. Although it should fact, MC can be highly advantageous to directly visualize the be theoretically inherited by an autosomal dominant 4q35 and 10q26 loci and discriminate among 4qA and other mode, it is not so frequent in the clinical practice. In our alleles. In addition, this approach may help to provide an ac- experience, molecular confirmation for FSHD1 is positive curate sizing of D4Z4 array and, subsequently, correlate it with in about 60% of subjects with a clinical suspect of dis- FSHD-related clinical features. However, the expensiveness, ease. This apparent low detection rate of the molecular the need for further automation, and quicker analytical times analysis is explained by the clinical variability of the phe- hinder the replacement of Southern blotting with MC for notype that involves just a hyperCKemia (high levels of D4Z4 sizing [5]. Concerning nanochannel-based optical map- blood creatine kinase, CK) with poor clinical signs, in ping approach, it proved to be able to provide an accurate most cases. It is important to remind that CK is a meta- quantification of the number of D4Z4 repeats, discriminate bolic enzyme that is highly expressed in cardiac and 62 Neurogenetics (2019) 20:57–64 skeletal muscles and is detected in blood in response to Funding information This work is funded by the Italian Ministry of muscle injury or dystrophy. However, it cannot be utilized Health (5X1000-2016 and 5x2017 MINSAL.3). as an accurate diagnostic marker for FSHD, since it is Compliance with ethical standards highly variable among man and woman and appears slightly/moderately increased in FSHD [4]. Some people Conflict of interest The authors declare that they have no conflict of do not show any symptoms during their life despite car- interest. rying the genetic defect; others are only minimally affected or show non-muscular problems. What is surprising is that this variability exists also within the same family and References involves not only the severity, but also the age of onset of the disease. In addition, we observed that as many as 2% 1. 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