Type 2 diabetes (T2D) is a major health priority worldwide and the majority of people with diabetes live with multimorbidity (MM) (the co-occurrence of ≥2 chronic conditions). The aim of this systematic review was to explore the association between MM and all-cause mortality and glycaemic outcomes in people with T2D.The search strategy centred on: T2D, MM, comorbidity, mortality and glycaemia. Databases searched: MEDLINE, EMBASE, CINAHL Complete, The Cochrane Library, and SCOPUS. Restrictions included: English language, quantitative empirical studies. Two reviewers independently carried out: abstract and full text screening, data extraction, and quality appraisal. Disagreements adjudicated by a third reviewer.Of the 4882 papers identified; 41 met inclusion criteria. The outcome was all-cause mortality in 16 studies, glycaemia in 24 studies and both outcomes in one study. There were 28 longitudinal cohort studies and 13 cross-sectional studies, with the number of participants ranging from 96–892,223. Included studies were conducted in high or upper-middle-income countries. Fifteen of 17 studies showed a statistically significant association between increasing MM and higher mortality. Ten of 14 studies showed no significant associations between MM and HbA1c. Four of 14 studies found higher levels of MM associated with higher HbA1c. Increasing MM was significantly associated with hypoglycaemia in 9/10 studies. There was no significant association between MM and fasting glucose (one study). No studies explored effects on glycaemic variability.This review demonstrates that MM in T2D is associated with higher mortality and hypoglycaemia, whilst evidence regarding the association with other measures of glycaemic control is mixed. The current single disease focused approach to management of T2D seems inappropriate. Our findings highlight the need for clinical guidelines to support a holistic approach to the complex care needs of those with T2D and MM, accounting for the various conditions that people with T2D may be living with.International Prospective Register of Systematic Reviews CRD42017079500 Type 2 diabetes (T2D) is a leading health priority. Over 424 million people live with diabetes worldwide, leading to $727 billion US dollars in healthcare expenditure . It is eT2D management is complex and requires continuous efforts from both clinicians and patients to implement recommendations for self-management and pharmacotherapy to achieve evidence-based targets. For patients who have other chronic conditions in addition to T2D, this complexity is amplified. This is important as T2D rarely occurs on its own–multimorbidity (MM) (presence of ≥ 2 conditions) is the norm, with approximately 85% of those living with T2D having at least one other chronic condition [MM produces many challenges. It is associated with lower quality of life, increased costs, a reduced ability to make lifestyle changes and may be associated with complex therapeutic regimens , increasWhile MM brings about multiple challenges in a clinical sense, it also presents multiple considerations from a conceptual perspective. The terms comorbidity and MM are often used interchangeably . Only moDespite MM presenting many challenges, there is no “gold standard” for the measurement of MM . We stilMM is common in T2D and brings many challenges, yet currently little is understood about the association between MM and outcomes in T2D. This systematic review seeks to explore literature on the association between MM condition count in people with T2D and the following two primary outcomes: 1) all-cause mortality; and 2) glycaemia (measured by HbA1c). Secondary outcomes of interest include other measures of glycaemia: 1) hypoglycaemia, 2) hyperglycaemia; and 3) glycaemic variability.Our detailed review protocol has been published elsewhere  and is sWe have followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines  and our th July 2017.A comprehensive search strategy was used to identify empirical quantitative studies with data on associations between MM condition count and our outcomes of interest in people with T2D. Target studies were observational studies that used either a cross-sectional or longitudinal cohort design. The search was limited to papers published in English but there was no restriction on publication date. A formal database search strategy was developed in consultation with a librarian (PC) from a biomedical library and members of our author group with expertise in MM and T2D, using a combination of free text search and medical subject headings; this is shown in We included empirical quantitative studies that included data on associations between MM condition count and adults with T2D and all-cause mortality or glycaemic outcomes. Full details of inclusion and exclusion criteria for studies are shown in The data screening process was conducted in three stages. Titles of identified studies in the searches were screened by the primary researcher (JC) with a deliberately inclusive approach to reduce the risk of missing potentially relevant studies. Abstract, full paper screening, data extraction and quality appraisal were undertaken by two individual reviewers with any inter-reviewer disagreements resolved by a third reviewer. Covidence , a systethe selection of the study groups; the comparability of the groups; and the ascertainment of the outcome of interest. We adapted the quality assessment scale to suit our systematic review; shown in Quality appraisal was conducted by two reviewers independently. All studies were assessed using the Newcastle-Ottawa quality assessment scale  which waIn total, 4,882 papers were identified with our search strategy, and 41 subsequently met our inclusion criteria. We included 41 studies in our review. Twenty-four studies –40 had gKey descriptive information of the included studies is summarised in The measure of MM condition count used in the included studies varied considerably. The majority of studies (24/41 59%)) , 55, 57 %) , 55, Only 11/41 27%) , 54, 57 % , 54, 5Description of the demographics of patients included in the studies is summarised in comparability domain, only 10 (24%) of the 41 included studies considered both age and known duration of diabetes in their data analyses.The quality appraisal of the included studies is summarised in We identified 17/41 studies  that incWe identified 25 studies –40, 57 tThe majority of studies (10/14), 35, 38 An increase in MM count was significantly associated with hypoglycaemia in nine of the 10 included studies that presented hypoglycaemia as a glycaemic outcome , 40, 57.One study explored the relationship between MM count and glycaemia as a continuous measure of fasting plasma glucose . The stuNo study reported glycaemic variability as an outcome measure when exploring the relationship between MM count and glycaemia.Heterogeneity of the included studies can be seen in multiple aspects. It arises from the multiple MM measures used in the included studies, the treatment of MM in data analysis either as continuous or categorical variables and the different statistical analyses used. This is summarised in Figs To the best of our knowledge, this is the first systematic review to synthesise the existing evidence on associations between MM, all-cause mortality and glycaemia in people with T2D.The key findings from our review were that increased MM condition count in those with T2D is significantly associated with all-cause mortality and hypoglycaemia. However, the evidence of effects on other measures of glycaemia including HbA1c and fasting plasma glucose is mixed. No studies explored the associations of MM with glycaemic variability.Our findings are consistent with previous literature where increasing MM has been shown to be associated with increased mortality in people with T2D –60. Our We found no convincing evidence of an association between MM count and HbA1c. This was unexpected as it has been well-established that HbA1c is an important clinical outcome to consider in people with T2D and glycaemic management is a key component of clinical guidelines for T2D. This suggests that the association of MM with mortality that we identified is not necessarily strongly mediated by glycaemia. Studies that have explored the relationship between HbA1c and mortality showed conflicting results where some studies suggested that increased HbA1c was significantly associated with increased mortality , 62 howeWe found a significant association between MM and hypoglycaemia with 9/10 studies demonstrating this. Previous studies have suggested that the presence of coexisting conditions may increase a person’s vulnerability to both adverse clinical outcomes, including death, and severe hypoglycaemia , 67. ThiAnother key finding is that despite our comprehensive search of the literature, there was no study that explored the effects of MM count on glycaemic variability. Glycaemic variability, which refers to fluctuations in blood  glucose levels, is a relatively new measure of glycaemia in people with T2D. There is growing interest in targeting reduced glycaemic variability as an independent clinical goal because higher glucose variability is thought to be associated with the development of chronic diabetes complications . It is cet al, however, demonstrated that using a condition count was not inferior to the more complex CCI (which applies weightings to the count) when predicting mortality in people with T2D [The studies included in our review were highly heterogenous, particularly in the way that MM was measured. There is no consensus as to what is the best method to measure MM, and which is most appropriate to use to predict mortality and other clinical outcomes. Monami with T2D . The attwith T2D  and arguwith T2D .Key strengths of our review are our adherence to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines, our comprehensive search strategy, and that all screening and data extraction were performed by two reviewers independently.We restricted our search to studies in the English language, which might be viewed as a limitation, although there is increasing evidence that this is unlikely to be a particular problem . We did Important strengths of our review are that we conducted an exhaustive search of five electronic databases and our tight inclusion and exclusion criteria allowed us to gather any data that explored associations between MM and our outcomes of interest, even if the study did not specifically aim to explore this relationship.A major advantage of our review is that it brings together information about the effects of MM in people with T2D from various sources to create a comprehensive picture of its effects on a number of outcomes. However, one limitation is that the included studies and therefore study participants were highly heterogenous, making comparisons between studies difficult, which prevented meta-analyses and limited us to a narrative synthesis. The large variation arose from the many ways MM is defined, the way outcomes are reported and the varying methods of statistical analyses. Another limitation of our review is that we did not explore the effects of specific multimorbid conditions on our outcomes of interest. While specific comorbidities such as diabetic nephropathy may potentially have contributed to hypoglycaemia as an endpoint in the included studies we were not able to explore this. Our focus is on the overall burden of illness experienced by people with T2D in the form of a MM count. The impact of specific conditions on outcomes in people with MM could be explored in future studies.We have reviewed the existing literature to provide a comprehensive summary of the effects of MM count in people with T2D on all-cause mortality and glycaemic outcomes. Our findings show that MM is significantly associated with increased mortality and hypoglycaemia. However, the effects of MM on other measures of glycaemic control, particularly HbA1c, is mixed.Our review findings emphasise the need for clinical guidelines and clinicians to support a holistic approach to the complex care needs of those with T2D living MM, where care of the whole person should be the primary concern, an approach that accounts for the full range of conditions that people with T2D may be living with.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOC)Click here for additional data file.


This article is an update of the best practice guidelines for the molecular analysis of Prader-Willi and Angelman syndromes published in 2010 in BMC Medical Genetics [ Prader-Willi syndrome PWS, #176270) is characterised by severe hypotonia and feeding difficulties in early infancy, followed in later infancy or early childhood by excessive eating and gradual development of morbid obesity (unless eating is controlled by dietary restriction or behaviour modification). Motor milestones and language development are delayed. All individuals have some degree of cognitive impairment, although some will have an IQ within the normal range. A distinctive behavioural phenotype  is common. Hypogonadism is present in both males and females, and manifests as genital hypoplasia, incomplete pubertal development, and in most, infertility. Short stature is common; characteristic facial features, strabismus, and scoliosis are often present, and non-insulin-dependent diabetes mellitus often occurs in obese individuals. Consensus diagnostic clinical criteria for PWS have been developed 70 is cha, but genAngelman syndrome AS, #105830) is characterised by severe developmental delay, absent or severely limited speech, gait ataxia and/or tremulousness of the limbs, and a unique behaviour with a happy demeanour that includes frequent and sometimes inappropriate laughter, smiling, and excitability. In addition, microcephaly and seizures are common. Affected individuals usually have a characteristic electroencephalography (EEG) appearance with striking high-voltage activity. Developmental delay is first noted at around 6 months of age; however, the unique clinical features of AS may not manifest until after one year of age, and it can take several years before the correct clinical diagnosis is obvious. Consensus clinical diagnostic criteria have also been developed for AS , 5. Agai830 is chMKRN3, MAGEL2, NDN, PWRN1, NPAP1 (previously C15orf2), SNURF-SNRPN and several C/D box small nucleolar RNA (snoRNA) genes  contains a cluster of imprinted genes, which are under the control of an imprinting centre (IC) . Some of:TSS-DMR ). The SNSNORD64, SNORD107, SNORD108, SNORD109A and SNORD109B), the two snoRNA genes SNORD116 and SNORD115 are present in 24 and 47 gene copies, respectively. It has been demonstrated that deficiency of SNORD116 snoRNAs causes the key characteristics of the PWS phenotype [Whereas most of the snoRNA genes are present as single genes  cause AS [UBE3A expression is restricted to brain cells where expression is exclusively from the maternal chromosome and disruption of expression of this gene is the major cause of the disease. In contrast to PWS, ~15% of patients with the clinical diagnosis of AS have a genetic defect of unknown aetiology.In addition, disease causing variants (point mutations) in the imprinted cause AS –20. ImprThe IC has been defined using microdeletions in a small number of patients with PWS or AS and an ID, who have small, atypical deletions. It has a bipartite structure with two critical regions, the AS-SRO  and the PWS-SRO (PWS-IC element ). By anaA summary of the causative genetic mechanisms and recurrence risks in PWS and AS is given in Tables UBE3A sequence analysis detects disease causing variants in approximately a further 5–10% of individuals with AS, however UBE3A analysis is not considered further in this article.There are a number of molecular genetic approaches to confirm these two disorders. The most common is DNA-based methylation testing to detect hyper- or hypomethylation within the PWS and AS critical region. This will detect more than 99% of individuals with PWS and ~80% of individuals with AS. The original guidelines for PWS and AS were assessed by consideration of the external quality assessment returns submitted to the European Molecular Genetics Quality Network (EMQN) and the United Kingdom External Quality Assessment Scheme (UK NEQAS) for Molecular Genetics over a 5-year period. The guidelines were posted on the web-site of the UK Clinical Molecular Genetics Society  since 2016) for consultation and amendment between 19 May 2008 and 6 January 2010 and heads of the constituent laboratories were invited to comment. In the light of feedback amendments were made and the final document was ratified by the CMGS Executive Committee on 10 January 2010. In addition they were approved by the European Molecular Genetics Quality Network (EMQN) Steering Group on 22 January 2010.The updated version in this article was available through the EMQN for consultation and amendment by the community of 105 laboratories participating in the EMQN-organised scheme for PW/AS, between 20 June 2018 and 13 July 2018. The feedback was collected, evaluated and the draft document amended accordingly.(i)The simultaneous assessment of methylation status and genomic dosage at numerous sites across the 15q11q13 region, by the use of methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA). This approach will confirm the diagnosis and further identify the presence of a causative deletion (large deletion or deletion of the imprinting centre (IC-deletion)). In the absence of a deletion methylation results alone are not sufficient to discriminate between UPD and ID. Therefore, microsatellite analysis is required.(ii)SNRPN locus by use of methylation-specific PCR (MS-PCR). This approach will confirm a diagnosis but will provide no further information regarding the disease mechanism. Thus, it is still open whether the molecular cause is a large deletion, a UPD or an ID with or without an IC-deletion necessitating follow up studies ).The detection of methylation status solely at the The approach to the laboratory diagnosis of PWS and AS depends on many factors, including the availability of samples, the arrangement of laboratory services and the patterns of referral. The most sensitive single approach to diagnosing PWS and AS is to study the methylation within 15q11q13 using molecular genetic techniques. These will detect deletions, UPD and ID by establishing either hypermethylation/complete methylation (PWS) or hypomethylation/complete absence of methylation (AS). Note that for AS mosaic cases only a partial hypomethylation is detected. Broadly speaking, methylation studies take one of two forms:It is essential to note that approach (ii) will not distinguish between the molecular causes. MS-MLPA however provides more information in this regard (see below). Figure http://www.mrc-holland.com). The MS-MLPA kit contains copy number probes, specific for sequences in or close to the PWS/AS critical region on 15q11q13 as well as outside the PWS/AS region and on other chromosomes which serve as controls. Among the PWS/AS specific probes, there are methylation-sensitive probes that contain a HhaI restriction site. The current MS-MLPA kit ME028-C1 contains five methylation-sensitive probes from the PWS/AS region that represent differentially methylated sites (four for the SNURF-SNRPN exon1/promoter region and intron 1 and one for MAGEL2), and another one from the completely unmethylated promoter region of UBE3A. Moreover, the kit contains additional methylation-sensitive control probes for completely unmethylated sequences from other chromosomes that will indicate complete digestion by the HhaI enzyme and therefore serve as digestion control probes.MS-MLPA provides a means to simultaneously detect copy number changes and DNA methylation within 15q11q13 in a semi-quantitative manner. Almost all laboratories using this approach make use of a commercially available MS-MLPA kit available from MRC Holland  and an IC-deletion. Furthermore, there are two probes for SNRPN exon U5 which can be used to detect IC-deletions in patients with AS and an ID. Both lie in the smallest region of deletion overlap found in patients with AS and an imprinting defect (AS-SRO). Beside IC-deletions, small deletions affecting the SNORD116 gene region or partly the UBE3A gene can be detected as well. For UBE3A deletion detection another MLPA kit  is available that covers each exon. But this is not the subject of these guidelines.Dosage analysis by MS-MLPA offers the opportunity to detect large deletions, the most frequent molecular lesions in patients with PWS and AS. In rare cases, a larger deletion can extend telomeric. Another advantage of the MS-MLPA kit is that it will identify IC-deletions in PWS and AS cases with an ID. The kit ME028-C1 contains two probes for the MS-MLPA has become the method of choice in many diagnostic laboratories as it investigates methylation status of single CpGs at several positions inside a DMR, thereby reducing the risk of false-positive or false-negative results due to SNPs or technical problems. Whereas the MS-PCR is restricted to the CpGs at the primer binding site(s).(i)NIPA1 and TUBGCP5, copy number variation has been observed. This has been attributed to deletions and duplications encompassing these two probes, and complicates the definition of class I and class II deletions.For the two most centromeric probes, The frequency of the BP1-BP2 duplication seems to be around 0.5% in the general population with the deletion being only slightly less frequent .The phenotypic outcome of these copy number variations is still a matter of debate and especially the deletion has been linked to a wide variety of phenotypic features from developmental delay to congenital heart defects –26. NeweSNRPN exon u1B probe.Variation in the hybridisation efficiency due to a copy number variation is also observed for the (ii)SNPs under hybridisation binding sites can influence probe signals. Consequently, laboratories must exercise extreme caution when interpreting results from a single probe.(iii)MAGEL2 locus is frequently observed.Normal variation in the degree of methylation at the There appears to be some naturally-occurring variation in dosage and methylation status which must be taken into account when interpreting MS-MLPA results:MAGEL2 and NDN locus  within the primer binding sites  remains unknown. Consequently, patients who were found to have a deletion or an isodisomy and also patients without evidence for a deletion must be investigated for methylation.When a molecular diagnosis of AS or PWS is confirmed with any of the above techniques microsatellite analysis will often be required to distinguish between a UPD and an ID. In some cases MSA is also used to detect large deletions, if enough informative markers are present. However we would recommend dosage analysis by MLPA as this can immediately detect large and IC-deletions.D15S113. This is a CA repeat from within the critical region. The presence of null alleles  has been observed with this marker and can complicate the analysis of AS and PWS cases. Under certain conditions, a non-amplification allele can be misinterpreted as a small deletion. The frequency of these alleles in families without AS has been estimated to be around 4%. Alternative primers can be designed; however, this marker is best avoided.D15S817. The presence of three alleles has been observed with this marker with certain primer sets due to complex duplications in the region where the marker is located. Since there is a low frequency/density of useful markers for the more centromeric PWS/AS region, the use of the following primer pair  can resolve this problem.At present many microsatellites are available for chromosome 15, and it is outside the scope of this article to provide a comprehensive list of suitable markers. However, it is worth noting the following markers that have been used in the past and are known to be compromised:For microsatellite analysis we recommend choosing markers within and outside the PWS/AS region and analysing an appropriate number of markers to be able to make a solid interpretation. In some countries it is compulsory to analyse at least eight markers. Moreover, an interpretation should never be built on one single informative marker.Uniparental inheritance both inside and outside the critical region: In this case, the disease is due to UPD. It is important to note that AS and PWS can be caused by either chromosomal isodisomy or heterodisomy (Meiosis I or Meiosis II non-disjunction). Further, heterodisomy or isodisomy at a single locus does not necessarily reflect the disomy status along the entire chromosome depending on the rate and level of crossing over and the meiotic stage at which non-segregation occurred.Biparental inheritance both inside and outside the critical region: In this case the disease is due to an ID. Exclusion of an IC-deletion must ensue if not already performed (e.g. by MS-MLPA).Uniparental inheritance inside the critical region, biparental outside: In this case the disease is most likely due to a deletion of the critical region. Laboratories must not interpret results from a single informative microsatellite without supporting evidence. In very rare cases such a constellation can be seen in patients with a segmental UPD [ntal UPD .Once the diagnosis of PWS or AS has been confirmed using methylation analysis, the interpretation of the microsatellite results is as follows:A normal methylation pattern rules out PWS on the basis of most known cases to date (sensitivity ~99%) and around 80% of AS cases.Despite early reports of possible deletion mosaicism in PWS , 37 the UBE3A analysis as mutations in this gene will have a recurrence risk of 50% if the mother is a non-mosaic mutation carrier.In the case of AS, if the clinical suspicion remains strong with normal methylation then it is recommended to undertake OCA2 gene . In class II deletions (60–70% of deletion cases) the proximal breakpoint is between D15S541 and D15S543 . The clustering of the deletion breakpoints is due to the presence of large duplicated sequence stretches of 200–400 kb in size in the common breakpoint regions that are susceptible to non-homologous crossovers [The de novo interstitial deletion of chromosome 15, del(15)(q11q13), which includes the entire imprinted domain plus several non-imprinted genes, extends ~5–7 Mb and is found in the majority of patients with PWS and AS. In both syndromes, the same region is affected, but in PWS the deletion is always on the paternal chromosome, whereas in AS it is always on the maternal chromosome. This deletion is probably one of the most common pathogenic deletions observed in humans [ossovers –41. Therossovers . Cases oUBE3A is silent on the paternal chromosome, so upd(15)pat is associated with a complete loss of function of this gene in this tissue. UPD can arise as a result of meiotic and/or mitotic errors [n = 46) is reduced to a haploid set (n = 23). Nondisjunction of the homologous chromosomes 15 during, e.g., female meiosis I or nondisjunction of the two sister chromatids during female meiosis II results in an oocyte with two chromosomes 15 or no chromosome 15. In these cases, fertilisation by a sperm with one chromosome 15 will result in a zygote which is trisomic or monosomic for chromosome 15 respectively. These conditions are not compatible with normal development, but can be ‘rescued’ by loss of one chromosome 15 from a trisomic cell or duplication of the paternal chromosome 15 in a monosomic cell. With trisomy rescue, in two-thirds of cases, one of the two maternal chromosomes will be lost from the trisomic cell, resulting in a normal set of chromosomes. If, however, the paternal chromosome is lost, the cell is left with two maternal chromosomes 15 (upd(15)mat; PWS). Duplication of the paternal chromosome 15 in a monosomic cell will lead to upd(15)pat (AS).The second most frequent finding in PWS is maternal UPD (upd(15)mat; nomenclature according to ISCN 2016 ) of chroc errors . MechaniAlternative mechanisms for UPD such as complementation involving both a nullisomic and disomic gamete or rescued paternal 15 trisomy have also been reported, however are considered rare .Cases of UPD should be further investigated by cytogenetic analysis to rule out the presence of a (very rare) balanced Robertsonian translocation in one of the parents - both parents have to be analysed irrespective of a detected upd(15)mat or upd(15)pat.There are a small number of patients with either PWS (1%) or AS (3%) that show a hypermethylation/complete methylation or hypomethylation/complete absence of methylation, respectively, without a large deletion of 15q11q13. Moreover, they show biparental inheritance for chromosome 15 markers, both inside and outside the critical region. These patients have an ID. The majority of these patients are sporadic cases without any detectable deletion or a DNA sequence change at the IC region. However, in 10–15% of cases, the ID is caused by an IC-deletion. In most cases, the IC-deletion is familial and thus associated with a 50% recurrence risk. In some cases the IC-deletion is de novo or a consequence of germ line mosaicism in the father or the mother. In these families, the recurrence risk ranges from 0–50%, depending on the degree of the mosaicism in the germ line . An IC-dIf a laboratory does not use an assay capable of assessing copy number in the AS- and PWS-SROs then, in the case of a confirmed diagnosis where a common deletion or UPD has been excluded, it is essential that the family workup is completed in a laboratory able to undertake this test. On confirmation of an IC-deletion parents should also be tested if available (father in case of PWS and mother in case of AS) to establish recurrence risks in the family.As mentioned before, more than 40% of AS imprinting defect patients without an IC-deletion have a partial hypomethylation and therefore are somatic mosaics for an ID (the detected degree of hypomethylation depends on the number of cells carrying the ID). In these cases the ID occurred after fertilisation and is not associated with an increased recurrence risk .It is recommended that laboratories do not use joint PWS/AS report templates.It is essential to state clearly on the report the method(s) used to carry out genetic analysis, with an appropriate reference; for example, “ME028-C1 (MRC Holland)”, always including the version number or “Zeschnigk et al. 1997 Eur J Hum Genet, 5:94-99”.Identifier of the patient and of all other family members from whom a sample was used for diagnostics, e.g. in MSA, must be given.The source of the DNA sample should be given on the report, as it can be important for example in case of prenatal diagnosis.Results should be reported in a clear and concise fashion. The interpretation should be given separately and not be merged with the results.It should be stated clearly that ‘the diagnosis of PWS or AS is confirmed’. Phrasings like ‘is consistent with’, ‘supports the diagnosis’, ‘is in accordance with’, ‘are compatible with’ are ambiguous.Reports should inform about the recurrence risk.Genetic counselling should be offered to all families irrespective of whether a diagnosis is confirmed or not (different standards may apply in different countries).Limitations and test sensitivity should be mentioned if diagnosis could not be confirmed (e.g. low grade mosaicism might escape detection).Inadequate terms to describe methylation results should be avoided. For the description of the result obtained terms as ‘PWS pattern’, ‘abnormal methylation pattern’, ‘change in methylation’ or ‘pathological result’ are not appropriate. The best is to describe what is observed. Examples for such descriptions including adequate terms of the methylation result are given below.Some general considerations for the reports:More specific recommendations are as follows:(i)Normal methylation and normal gene dosage at 15q11q13 detected:This result excludes a large deletion on the paternal allele, a UPD of chromosome 15 and an ID. A diagnosis of PWS is highly unlikely (sensitivity of the test ~99%).(ii)Heterozygous deletion and hypermethylation/complete methylation detected:TUBGCP5 to OCA2, class I deletion; 31 of probes from MKRN3 to OCA2, class II deletion; number of probes depending on the kit version used, here ME028-C1). The methylation analysis shows a hypermethylation/complete methylation at all five methylation-sensitive probes, indicating that the deletion is on the paternal allele.Gene dosage analysis shows a 50% reduction for # probes within the chromosomal region 15q11q13 (33 of probes from This result confirms the diagnosis of PWS. The molecular cause is a heterozygous deletion within 15q11q13 (class I or class II deletion) on the paternal allele.Cytogenetic analysis in the patient and the father should be performed to rule out the presence of cytogenetic rearrangements which would increase the recurrence risk.(iii)Normal gene dosage and hypermethylation/complete methylation detected:Normal gene dosage, but a hypermethylation/complete methylation at all five methylation-specific probes. This result confirms the diagnosis of PWS. The molecular cause of PWS is either a maternal UPD of chromosome 15 or an ID.Microsatellite analysis should be performed in the family to differentiate between a upd(15)mat and an ID.If a upd(15)mat is confirmed subsequent cytogenetic analyses should be performed in the patient and the parents to rule out cytogenetic rearrangements which would increase the recurrence risk.If an ID is confirmed the recurrence risk is not increased, since an IC-deletion has been excluded.(iv)Heterozygous deletion of the IC and hypermethylation/complete methylation detected:Gene dosage analysis shows a 50% reduction for # probes located in the IC (number and names of probes according to the kit version used). The methylation analysis shows a hypermethylation/complete methylation at all five methylation-specific probes, indicating that the IC-deletion is on the paternal allele.This result confirms the diagnosis of PWS. The molecular cause of PWS is an ID due to an IC-deletion.MS-MLPA analysis in the father should be recommended to estimate the recurrence risk. If the father is a non-mosaic carrier of the IC-deletion the recurrence risk is 50%. If he has no detectable deletion in blood DNA this does not exclude germ line mosaicism, which is also associated with an increased recurrence risk (see section “Imprinting defect”).If appropriate other family members should be investigated, too.(i)Normal methylation at 15q11q13 detected:This result excludes a large deletion on the paternal 15q11q13 allele, a UPD of chromosome 15 and an ID. A diagnosis of PWS is highly unlikely (sensitivity of the test ~99%).(ii)Hypermethylation/complete methylation detected:No paternal, unmethylated band detected in the methylation analysis at 15q11q13. This result confirms the diagnosis of PWS. The underlying molecular cause can be either a large deletion of 15q11q13 on the paternal allele, a maternal UPD of chromosome 15 or an ID (with or without an IC-deletion). Further molecular genetic testing  is necessary.As MS-PCR or equivalent methods that investigate only the methylation status cannot differentiate between the possible molecular genetic mechanisms underlying the PWS diagnosis, additional investigations regarding gene dosage and segregation of chromosome 15 have to be requested. This is of utmost importance as the recurrence risk cannot be determined without knowledge of the underlying molecular genetic cause.(i)Normal methylation and normal gene dosage at 15q11q13 detected:This result excludes a large deletion on the maternal allele, a UPD of chromosome 15 and an ID.UBE3A should ensue if the clinical re-assessment of the patient still suggests AS.This result does not exclude a diagnosis of AS. Analysis of (ii)Heterozygous deletion and hypomethylation/complete absence of methylation detected:TUBGCP5 to OCA2, class I; 31 of probes from MKRN3 to OCA2, class II; number of probes depending on the kit version used, here ME028-C1). The methylation analysis shows a hypomethylation/complete absence of methylation at all five methylation-specific probes, indicating that the deletion is on the maternal allele.Gene dosage analysis shows a 50% reduction for # probes within the chromosomal region 15q11q13 (33 of probes from This result confirms the diagnosis of AS. The molecular cause is a heterozygous deletion within 15q11q13 (class I or class II deletion) on the maternal allele.Cytogenetic analysis in the patient and the mother should be performed to rule out the presence of cytogenetic rearrangements which would increase the recurrence risk.(iii)Normal gene dosage and hypomethylation/complete absence of methylation detected:Normal gene dosage, but a hypomethylation/complete absence of methylation at all five methylation-specific probes. This result confirms the diagnosis of AS. The molecular cause of AS is either a paternal UPD of chromosome 15 or an ID.Microsatellite analysis should be performed in the family to differentiate between a upd(15)pat and an ID.If a upd(15)pat is confirmed subsequent cytogenetic analyses should be performed in the patient and the parents to rule out cytogenetic rearrangements which would increase the recurrence risk.If an ID is confirmed the recurrence risk is not increased, since an IC-deletion has been excluded.(iv)Normal gene dosage and partial hypomethylation/partial absence of methylation detected:Normal gene dosage, but a partial hypomethylation/partial absence of methylation at all five methylation-specific probes. This result confirms the diagnosis of AS. The partial hypomethylation/partial absence of methylation indicates that a somatic mosaic is present where cells with a normal imprint and cells with either a paternal UPD of chromosome 15 or an ID are present.Microsatellite analysis should be performed in the family to differentiate between these two possibilities.(v)Heterozygous deletion of the IC and hypomethylation/complete absence of methylation detected:Gene dosage analysis shows a 50% reduction for # probes located in the IC (number and names of probes according to the kit version used). The methylation analysis shows a hypomethylation/complete absence of methylation at all five methylation-specific probes, indicating that the IC-deletion is on the maternal allele.This result confirms the diagnosis of AS. The molecular cause of AS is an ID due to an IC-deletion.MS-MLPA analysis in the mother should be performed to estimate the recurrence risk. If the mother is a non-mosaic carrier of the IC-deletion the recurrence risk is 50%. However, an absence of the deletion in blood does not exclude a germ line mosaicism which is associated with an increased recurrence risk (see section “Imprinting defect”).If appropriate other family members should be investigated, too.(i)Normal methylation at 15q11q13 detected:This result excludes a large deletion on the maternal allele, a UPD of chromosome 15 and an ID.UBE3A should be performed if the clinical re-assessment of the patient still suggests AS.This result does not exclude a diagnosis of AS. Analysis of (ii)Hypomethylation/complete absence of methylation detected:No maternal, methylated band detected in the methylation analysis at 15q11q13. This result confirms the diagnosis of AS. The underlying molecular cause can be either a large deletion of 15q11q13 on the maternal allele, a paternal UPD of chromosome 15 or an ID with or without an IC-deletion. Further molecular genetic testing  is necessary.(iii)Partial hypomethylation/partial absence of methylation detected:A faint maternal, methylated band detected in the methylation analysis at 15q11q13. This result confirms the diagnosis of AS. The underlying molecular cause can be either a large deletion of 15q11q13 on the maternal allele, a paternal UPD of chromosome 15 or an ID each in a mosaic state. Most likely are a UPD and an ID. Further molecular genetic testing  is necessary.As MS-PCR or equivalent methods that investigate only the methylation status cannot differentiate between the possible molecular genetic mechanisms underlying the AS diagnosis, additional investigations regarding gene dosage and segregation of chromosome 15 have to be requested. This is of utmost importance as the recurrence risk cannot be determined without knowledge of the underlying molecular genetic cause.SNRPN exon 1 is established early in embryonic development and testing DNA extracted from both amniotic cells and chorionic villi (both native and cultured) has been shown to give reliable results [NDN locus should not be interpreted in case of prenatal testing. The methylation at the MAGEL2 locus is more similar to the methylation status at the SNRPN region than NDN, but may not always be fully established at time of sampling, and is therefore not reliable and should also not be interpreted in case of prenatal testing . Methylation status at  results . The metUBE3A in AS cases that are either de novo or a consequence of germ line mosaicism in the parent, the recurrence risk is difficult to predict but may be as high as 50%. In cases of sporadic ID with no detectable deletion, the recurrence risk appears to be low, however since the possibility of recurrence cannot be excluded a prenatal diagnosis should be offered.For cases of ID due to IC-deletions or disease causing variants in Diagnoses which need to be considered in infants with hypotonia include congenital myopathies, central and peripheral neuromuscular disorders, especially type 1 spinal muscular atrophy and the congenital form of myotonic dystrophy. Furthermore anomalies of the central nervous system and peroxisomal disorders should also be ruled out if chromosome 15 methylation is normal , 48.A further phenotype which presents with neonatal hypotonia and short stature together with later-onset obesity is Temple syndrome, an imprinting disorder which can be caused by deletions, ID and upd(14)mat involving the chromosomal region 14q32 , 50.When considering the differential diagnosis of older children with learning disability and obesity, Cohen syndrome, Borjesson-Forssman-Lehman syndrome , Bardet-Biedl syndrome, Alstrom syndrome and Fragile X syndrome along with chromosomal disorders including diploid/triploid mosaicism, 1p36 microdeletion syndrome and 16p11.2 deletion syndrome, should be considered . In manyMAGEL2 gene on 15q11q13 have been described in patients with Schaaf-Yang syndrome (#615547). The phenotypic overlap with PWS includes neonatal hypotonia, intellectual disability and later onset of obesity. Contractures of interphalangeal joints can serve as a distinguishing feature [Truncating disease causing variants of the imprinted rome #6157. The phdrome #6147. The p feature , 57.MECP2 gene located on chromosome Xq  [CDKL5 gene should be considered [ZEB2 gene on chromosome 2q, is associated with severe learning disability, limited speech, seizures and characteristic facial features that resemble those of AS. In addition Hirschsprung disease, congenital cardiac defects and agenesis of the corpus callosum may be associated with disease causing variants in ZEB2. A strong indication is the characteristic appearance of the ear lobes which are upturned and look like “shell pasta”. Pitt-Hopkins Syndrome (PHS) is a sporadic condition caused by disease causing variants or deletions of the TCF4 gene on chromosome 18q. Patients present with absent speech, seizures and facial features resembling AS, together with a sociable personality. The facial appearance in PHS coarsens with age and the lips in particular become prominent. Episodes of hyperventilation and apnoea may develop [CNTNAP2 and NRXN1. Breathing anomalies, epilepsy and autistic features are prominent features in these cases [SLC9A6 gene [MEF2C gene has been described due to larger deletions as well as disease causing variants in patients with intellectual disability, absence of speech and seizures. Additionally, stereotypic movements were present [Around 10–15% of patients with a clinical diagnosis of AS have no detectable disturbance at 15q11q13 using the techniques described here. In rare cases, these patients may be low grade mosaics for an imprinting defect. However, it is more likely that there is an alternative clinical diagnosis and a careful review of the patient’s history, clinical features and EEG findings is recommended. One diagnosis which should be considered in girls is Rett syndrome caused by haploinsufficiency of the ossible) , 59. It nsidered . Mowat-W develop , 62. Autse cases , 64. Ano9A6 gene , 66. Spe present , 68.MECP2 gene may also present with a phenotype suggestive of AS in males [MBD5 and a clinical and behavioural phenotype reminiscent of AS were reported [Several chromosome abnormalities have phenotypes that overlap with AS. The most common are the 1p36 subtelomeric deletion, a microdeletion of 17q21, and a terminal deletion of 22q13 , 70. Xq2in males . Profounin males . Severalreported . SeizureMTHFR) deficiency and adenylosuccinate lyase deficiency have been reported as presenting with learning disability, ataxia, seizures, autistic features and excessive laughter [Some rare metabolic disorders may present with AS-like symptoms. Methyltetrahydrofolate reductase , by achieving formal accreditation with a member organisation of the International Laboratory Accreditation Cooperation (ILAC) or equivalent national accreditation body.All tests should be validated/verified in individual laboratories prior to implementation; it is not acceptable to rely on the validation of a test by another laboratory, since that does not guarantee that it will perform accurately and reliably in all labs. A series of control samples representing all disease causing variant types should therefore be collected by each laboratory to facilitate test validation/verification, and exchange of samples between laboratories is encouraged to allow this. External quality assessment (EQA) schemes provide further validation of testing procedures and methods, and laboratories should participate annually in appropriate EQA schemes for PWS and AS testing. If this is not possible, inter-laboratory exchange of samples should be arranged to compare and validate test results.This is an update of the practical set of molecular genetic testing and reporting guidelines that have been developed for PWS and AS by Ramsden and colleagues . The updFeedback has been obtained from participants of the 2018 PWS/AS EMQN external quality assessment scheme (105 laboratories from 29 countries). Nine comments were received; most were minor largely typographic corrections and some points of clarity. There was no disagreement on the recommendations made. All comments have been incorporated into this final document.Supplementary Figure 1Supplementary Figure 2Supplementary information


DPYD gene can lead to significant toxicity associated with the administration of fluoropyrimidine chemotherapy.Dihydropyrimidine dehydrogenase deficiency secondary to polymorphisms in the DPYD gene was sequenced, and the patient was found to be heterozygous for three different polymorphisms that have reportedly been associated with dihydropyrimidine dehydrogenase deficiency.We report a case of a 59-year-old Lebanese woman with metastatic pancreatic cancer who received FOLFIRINOX therapy and developed severe 5-fluorouracil toxicity after a single cycle. The entire DPYD gene are very limited, we suggest that full gene sequencing should be carried out, at least in populations in which the allele frequencies are unknown.Because data regarding the prevalence and clinical significance of several heterozygous polymorphisms in a single  DPYD gene detected by sequencing the coding region and highly conserved exon-intron splice junctions, and we suggest that extended gene testing should be carried out, at least in populations in whom the allele frequencies are unknown.Fluoropyrimidine anticancer drugs such as 5-fluorouracil (5-FU) and capecitabine are commonly prescribed for adjuvant as well as palliative treatment of various solid malignancies. Treatment with these drugs is generally well tolerated, except in a small percentage of patients who develop severe, potentially life-threatening toxicity. The clinical presentation of this toxicity is similar to a 5-FU overdose and includes myelosuppression, mucositis, stomatitis, diarrhea, skin changes, and neurological abnormalities . Treatme2), 5-FU (2400 mg/m2), leucovorin (400 mg/m2), and oxaliplatin (85 mg/m2). She presented to the hospital 1 week after her first cycle with weight loss and decreased oral intake owing to odynophagia. She was diagnosed with grade 4 mucositis and was started on fluconazole and later acyclovir. Owing to very poor oral intake, total parenteral nutrition with electrolyte correction was necessary until the patient was able to better tolerate food. At presentation to the hospital, she also reported three or four episodes of watery bowel movements per day. All stool study results were negative, so she was considered to have grade 1 chemotherapy-induced diarrhea and was started on loperamide. On the second day of hospitalization, the patient developed febrile neutropenia, so piperacillin and tazobactam were initiated along with vancomycin. Subcutaneous filgrastim was administered daily for 9 days and then twice daily for 3 days until the neutropenia subsided. During her stay, the patient also developed a drop in hemoglobin and platelet count, as well as an erythematous rash over the trunk with desquamation of the skin under the breasts. Owing to the severe side effects, FOLFIRINOX was discontinued despite a decrease in tumor marker, and the protocol was changed to gemcitabine and nanoparticle albumin-bound paclitaxel with a good initial response to treatment. With most regimens for the treatment of advanced pancreatic cancer including 5-FU, its oral prodrug capecitabine, irinotecan, or oxaliplatin, and because of the severe reaction the patient experienced, we decided to test the patient for DPD and uridine diphosphoglucuronate-glucuronosyltransferase 1A1 (UGT1A1) deficiency. UGT1A1 deficiency is associated with Gilbert’s syndrome and increases the toxicity of irinotecan. Those two tests would help us determine which of the two drugs was the culprit and guide us to use either or both drugs in a reduced dose. The functional activity of the DPD enzyme was not measured, but the DPYD gene was analyzed by next-generation sequencing of both DNA strands of the entire coding region and highly conserved exon-intron splice junctions. The patient was found to have three different variations of the DPYD gene as follows: heterozygous pathogenic variant DPYD*2A , heterozygous deficiency-associated variant c.1601G>A (p.Ser534Asn) [cis or trans) could not be determined, because both parents were not available. The patient also had UGT1A1 deficiency and hence Gilbert’s syndrome.A 59-year-old Lebanese woman was started on the FOLFIRINOX chemotherapy protocol for metastatic pancreatic adenocarcinoma with irinotecan (180 mg/mr534Asn) , and hetr534Asn) , 12. AllDPYD gene is the most well-recognized cause of DPD deficiency, with the clinically most relevant polymorphism being DPYD*2A. The frequency of DPYD*2A is 1% to 2% in the Western world [DPYD*2A, only 25% of DPD-deficient patients are identified [DPYD*2A polymorphism is 0.6% in the Turkish population [DPYD*2A polymorphism or other variants have been reported in the Middle Eastern or Lebanese population. When we wanted to test the patient for DPYD deficiency, two options were available: either screen for the most common variants or sequence the coding region or highly conserved exon-intron splice junctions. One meta-analysis recommended testing for the DPYD variants IVS14+1G>A and 2846A>T [DPYD*2A polymorphism but also two other deficiency-associated variants that would have been missed if the entire coding region had not been sequenced. As far as we are aware, this is the first report of three pathogenic or deficiency-associated variants in the DPYD gene in the same patient. One study from the United Kingdom recently identified the c.1601G>A variant to be significantly associated with fluoropyrimidine toxicity [DPYD*2A variant may be sufficient to cause the toxicity seen, but we cannot be sure; perhaps the presence of the two other variants added to the severity of the toxicity. In addition, two of the three variants must occur on the same allele, and perhaps having two variants on the same allele contributed to a significant decrease in DPD activity. The patient received two other chemotherapeutic agents that might be partially responsible for the adverse events experienced. However, oxaliplatin toxicity generally manifests as neurological abnormalities along with liver toxicity [Genetic polymorphism in the rn world , and by pulation , but no  2846A>T , and ano 2846A>T . We electoxicity . In thistoxicity . As for toxicity . The facHad the variant DPYD*2A been tested alone and had it not been present, the toxicity would have been blamed on irinotecan alone, and 5-FU would have been used without reductions, which could have harmed the patient.DPYD gene in the same individual, full gene sequencing may be considered for all patients in the future.New technology applied to sequencing and bioinformatics is bringing down the costs of genetic testing. Because there are very limited data regarding the prevalence and clinical significance of several heterozygous polymorphisms of the


Management of trauma involves long-term bed rest even when muscle strength in the lower extremities is preserved. Prolonged bed rest reduces muscle mass and causes muscle atrophy. A recent study reported the efficacy of rehabilitation using electrical muscle stimulation (EMS) for muscle strength maintenance in intensive care unit patients with disturbance of consciousness. However, despite the expected benefits of EMS in maintaining muscle strength, little is known about its efficacy in trauma patients.A single-center, open-label, randomized controlled trial of 40 patients with pelvic fracture to test the effectiveness of 14 days of EMS. The primary outcome will be change in cross-sectional area of the thigh muscle between pre and post intervention, as measured on computed tomography images. We will analyze the primary endpoint by analysis of covariance (ANCOVA) and analyze the secondary endpoints in an exploratory manner.If our hypothesis is confirmed, this study will provide evidence that the use of EMS can be effective in preventing muscle atrophy.UMIN000030190. Registered on 1 December 2017.UMIN registration number: The online version of this article (10.1186/s13063-018-2872-4) contains supplementary material, which is available to authorized users. Management of trauma, such as cervical spinal cord injury and pelvic fracture, involves long-term bed rest even when muscle strength in the lower extremities is preserved. Prolonged bed rest reportedly reduces muscle mass by 6–40% . After jA recent study reported the efficacy of rehabilitation using electrical muscle stimulation (EMS) for muscle strength maintenance in intensive care unit patients with disturbance of consciousness , clinicaIn this study, we investigate whether EMS plus conventional physiotherapy is more effective than conventional physiotherapy alone for maintaining muscle strength or retarding its impairment in patients with pelvic fracture who are placed on bed rest.This trial is conducted at the Center Hospital of National Center for Global Health and Medicine.To investigate whether addition of EMS to conventional rehabilitation retards loss of muscle mass in trauma patients who require bed rest for at least 1 week despite no initial impairment in muscle strength in the lower extremities.Single-center, open-label, randomized controlled trial.Age 20–90 years, able to provide informed consent themselves or via a legal representativeTrauma inpatient with either unstable pelvic fracture or pelvic fracture at the site where load will be applied. Patients with pelvic fracture included those with an AO classification of 61B2.3 (open book), 61B3.1, 61B3.2, 61C2.1, 61C2.2, and 61C2.3, as well as those who were recommended to rest in bed in supine position for at least 1 week as determined by orthopedic surgeons that we consulted.Requires bed rest for at least 1 weekComputed tomography (CT) scans of pelvis and lower extremities taken at the time of hospital admissionPacemaker (contraindicated for use with EMS devices)History of neuromuscular disorder  and/or cerebral infarction Disturbance of consciousness impairing ability to follow instructionsBed-bound or wheelchair-bound, or with femoral neck fracture and/or intertrochanteric femoral fractureScore of 2 or more for intestinal or pancreatic trauma  )Fracture or skin injury (except for contusion) at the site for EMS application Inability to provide informed consentParticipation judged to be inappropriate by the physician in chargeRationale for criteria 1–2: use of EMS may aggravate preexisting conditions or evaluation may not be conducted accurately.For electrical muscle stimulation, we will use the AUTO Tens PRO Rehabili Unit B-SES  and G-TES  devices . We will wrap the AUTO Tens PRO belt electrodes around the abdomen, proximal aspect of the legs, and ankles for electrical stimulation of the entire lower extremities. Patients will receive one 20-min EMS session daily, consisting of 5-s stimulations (20 Hz) separated by 2-s rest intervals, for 5 days per week for 2 weeks. The output current will be in the range 2–15 mA, and the current will be increased to the maximum tolerable level within 3 days of starting rehabilitation. A co-investigator who is a physical medicine and rehabilitation physician will supervise the 14-day EMS protocol. In parallel, we will provide conventional physiotherapy on the same day as the EMS session . Conventional physiotherapy will consist of (1) hip and ankle joint mobility exercises and (2) lower limb-strengthening exercises in a supine position.Patients will receive the conventional physiotherapy protocol alone.Failure to elicit palpable muscle contraction at maximum tolerable current after three daily sessions due to patient discomfortAny injury to the skin and/or striated muscleVoluntary withdrawal of consent from the trialRationale for criteria 1 and 2: to eliminate the influence of these factors on the evaluation of efficacy and safety.Rationale for criterion 3: to respect participants’ voluntary decision.Change in cross-sectional area of thigh muscle between pre and post intervention, as measured on CT imagesChange in cross-sectional area of calf muscle between pre and post intervention, as measured on CT imagesReduction in thigh and calf circumference, measured at the same sites as CT image measurementsThigh and calf circumference measured on CT imagesChanges in manual muscle testing resultsWe will measure all secondary endpoints on the same dates that CT imaging is obtained.We will obtain baseline CT data of the region including the pelvis and lower extremities at the time of admission  and measure cross-sectional area of the thigh and calf muscles. Day 1 is defined as the first day of rehabilitation intervention.Thigh: height at the center of a line linking the greater trochanter and knee joint cleftCalf: height at the center of a line linking the fibular head and external condyleThigh and calf muscle cross-sectional area: we will extract areas with intensity in the range of 30 to 100 Hounsfield Units (HU) from CT slices and measure cross-sectional area to determine change between pre- and post-intervention valuesTo evaluate the outcomes of intervention, we will obtain post-intervention plain CT data of the region including the pelvis and lower extremities and measure cross-sectional area of the thigh and calf muscles on day 14 of intervention (or before the end of day 17). Specifically, we will obtain the following measurements.Aquilion™ CX TSX-101A/NA, Application Ver. V4.62JR019 SOMATOM Definition Flash VA44A, Somaris/7 Syngo CT 2012BWinNT 6.1, Service Pack 1, VA44A_08_P16 Aquilion ONE™ TSX-301A/2A, Application Ver. V4.7JR004 Discovery CT 750HD, Application Software, 11 MW44.11.V40_PS_HD64_G_GTL We will obtain CT scans in the supine position with both legs in the neutral position, with a pillow placed under both legs to avoid compression of the posterior aspect of the legs from the CT bed. We will ask patients to relax to minimize the influence of morphological changes due to muscle contraction. The following CT machines will be used:Target sample size: 50 patients (25 each in the intervention group and control group).In a previous study of disuse muscle atrophy in patients with consciousness disturbance in the intensive care unit (ICU) , change To calculate sample size for the present study, we determined that approximately 25 pelvic fracture patients required 2-week hospitalization at the Department of Emergency Medicine and Critical Care, Center Hospital of the National Center for Global and Medicine between 2015 and 2016, with no dropouts including those due to death. Thus, a total of 50 patients (25 in each group) is required for the present study.Patients who are deemed eligible for the study according to the inclusion and exclusion criteria will be enrolled in the study within 1–2 days after hospital admission. Before entry, patients themselves must sign informed consent forms. In the case that patients can follow instructions, but dementia or mental retardation makes the voluntary nature of their actions unclear, their legal representatives  must provide written informed consent.A computer-generated random number table will be prepared by an individual who is not an investigator. The table will be stored in a designated safe by the coordinator of the outpatient division of the Department of Emergency Medicine and Critical Care, who is not involved in the study. Study investigators who are the physician in charge of admitted patients satisfying the enrollment criteria will report the admissions to the coordinator. The coordinator will then allocate patients to either of the groups using the prepared random number table and will notify the investigators of the allocation results.Participant background: age, sex, medical history, height, weight, diagnosis at time of admission  and at the end of rehabilitation (days 14–17)Clinical testing: creatinine kinase (CK) level on days 0, 3, 7, and 14–17Functional Independence Measure score and Barthel ADL Index at 1 and 2 months after the injurySee Fig. For all participants who satisfy the inclusion criteria and start receiving the test intervention, as a definitive statistical analysis model, ANCOVA (regression model) was used to evaluate the effect of the treatment. The outcome measure was the rate of reduction, and independent variables included the treatment method, possible confounders in randomization, and baseline measure of muscle mass and analyze the secondary endpoints in an exploratory manner.While missing data were not dealt with in the present study, data may be complemented after some process as appropriate if there are a certain number of missing values.Assessor, participants, treatment team, and statisticians were unblinded.Conventional therapy will be continued in consultation with the physicians in charge.A monitoring officer will conduct monitoring before, during, and after completion, termination, or withdrawal from the study.Potential burdens and risksEMS is widely used in patients with cardiac disease or diabetes mellitus, and its sufficient safety has been proven. However, the adverse side effects listed below have been reported, and thus will be monitored for continuously during the study. If they do occur, the physician in charge will provide appropriate care and treatment.Skin injury and rash at the site of belt attachmentAggravation of pain at the site of fracturePain during EMS2.Expected benefitsThe benefits of conventional physiotherapy are already well established in rehabilitation. Also, EMS is reported to prevent muscle atrophy. Thus, addition of EMS to the conventional physiotherapy with proven benefits may enhance the therapeutic effect.Serious adverse eventsWe will record any adverse event listed below, occurring in the period between the date of providing informed consent and day 14, irrespective of the presence or absence of a causal relationship. Also, we will promptly report any occurrence to the head of the research institute via the reporting division of the hospital within 7 days after the serious adverse event is first noticed.Resulting in deathBeing life-threateningRequiring inpatient hospitalization or prolongation of existing hospitalizationResulting in persistent or significant disability/incapacityResulting in congenital anomalySerious adverse events:Other adverse eventsThe therapeutic devices that will be used in this study have already been approved and are routinely used in the clinical setting. Thus, we will not record non-serious adverse events.We will use the designated case report form to record case information. We will register the case information digitally, with the principal investigator assigning the case number. After registration, the principal investigator and an analyst will analyze individual cases. They will pass the analysis report to the planning and strategy manager.Access to the personal information and data collected in this study will be strictly limited to investigators of this study. The data collected will be for the purpose of conducting the study only. Investigators will handle personal information carefully. The principal investigator will put in place measures for appropriate information and data handlingPhysicians in charge will initially collect data on paper. They will then register the collected data in the study database. The study database will not contain personally identifiable informationThe study database will be stored on the hard disk drive of a password-protected computer kept in a lockable room. The study group will have a key to the room and maintain security of the computer password. The study group will appropriately handle research-related printed documents, memos, and other details, for example, by viewing them in an isolated room to avoid exposure to a third party and storing them in a lockable cabinet. The study group will minimize the use of portable electronic media, but if use is absolutely necessary, members must notify the principal investigator in advance of use and handle the media with extreme cautionThe principal investigator is responsible for storage of the anonymized random number table and for supervising the format. Once data are fixed in the study database, the principal investigator will hand the random number table to the center’s planning and strategy manager and no copies will be held by the study groupPersonal information and data collected during the study will be kept for 5 years from the time of publication of the main study outcomes before disposal. Both printed and electronic materials will be destroyed so that the data are unreadable before disposal. Some writable media may be reused after overwriting the original data with dummy data to make the original data irretrievableWhen disclosing study outcomes, study members will take all due care to prevent re-identification of de-identified dataThe study will be conducted after the study plan defining the study protocol is assessed and approved by the Ethics Committees of all participating centers. The Ethics Committees will review the study plan when protocol amendments are deemed necessary and must approve all protocol amendments.Study outcomes will be reported at professional meetings and submitted to a scientific journal for publication.Population aging is progressing worldwide, and the number of aged patients with fracture of the femur or pelvis due to falls or traffic accidents has been increasing. Muscle atrophy in patients with femoral fracture can be minimized by physiotherapy with a specific load early after operative fixation if performed soon after the accident. However, regardless of early fixation, patients with severe pelvic fracture may need long-term bed rest because a gravitational load cannot be applied for a relatively long time. This causes marked reduction of muscle mass of the lower extremities. EMS is applicable to such patients, and our study may provide evidence that EMS can prevent muscle atrophy to some extent and will provide an alternative to rehabilitation for these patients. Therefore, it is worthwhile to conduct this study.Finally, we discuss the potential limitations of the study. First, we think that differences among individuals may emerge when muscles contractions cause pain. To solve this problem, we will provide patients with sufficient analgesics, gradually increasing the dose as needed. A second limitation is that this study is open-labeled. We think that there is a possibility that patients of controls have slower rehabilitation than those of receiving EMS, and thus bias due to unblinding might persist. However, it is impossible to hide which allocated patients receive EMS because patients can feel when their muscles are stimulated


The aim of the present study is to assess the extent to which seafarers had been affected by severe mental stress situations and what possible psychological effects they may have.During the voyages of 22 German ships, a psychologically trained investigator interviewed 323 seafarers about severe mental stress or potentially traumatic events on board (participation rate 88.5%). Furthermore, a psychologist conducted semi-standardized interviews in 12 shipping companies and in 8 stations of the German Seafarers’ Mission on seafarers’ traumatic experiences.Of the seafarers surveyed on board, 116 (35.9%) experienced ship wrecks/severe accidents and 55 (17.0%) piracy on board. Experiences with stowaways were indicated by 126 seafarers (39.0%). Repeatedly having to unintentionally think about these events, being reminded of them by certain noises, smells, etc., or often dreaming of them were after-effects in 97 (83.6%) of the affected seafarers after ship wrecks/serious personal accidents on board and in 42 crew members (76.4%) after threats of piracy. Experiences of threat from stowaways had particularly affected non-European seafarers or ratings.According to the interviews with the shipping companies, a total of 14 deaths occurred in the last 3 years in the 12 interviewed shipping companies (due to heart attacks (3), severe cancer (3), very serious accidents (3), suicides (2) and 3 with unknown causes of death). In relation to the container ships of the investigated shipping companies, these frequencies correspond to a mortality rate of 78.4 per 100,000 seafarer years.According to the interviews in 8 Seafarers’ Missions, these organizations are in charge of emergency counselling in the form of crisis intervention when a seafarer has experienced serious psychological stress.A serious injury or even the death of a member is a serious psychological stress experience for the other crew members in the closed social system on board. These events are particularly distressing for Filipino seafarers with their strong sociocentric bonds and religious values. Priestly support is often required in order to stabilize the Filipino crew members.As a preventive measure, psychoeducation for seafarers should be established for coping with extreme mental stress situations. Within a small crew working together and living in a closed social system over a longer period of time, a serious injury or even the death of one crew member could cause a secondary mental trauma for the rest of the crew . Such trIn shipping, severe psychological stress or potentially traumatic events can result from ship wrecks and accidents, threats and experiences of violence from piracy, stowaways and in life-threatening rescue situations during the rescue of refugees on the high seas , 5.An own, so far unpublished analysis of sea accidents on ships in German territory revealed casualties on board 20 container ships in the period from 2005 to 2014. An investigation by the Federal Bureau of Maritime Casualty Investigation (BSU) (Maritime Safety Investigation -Law) identified accidents in which crew members were seriously or fatally injured, went overboard, experienced the total loss of their ship or were also responsible for it, as the major causes. However, there are also fatalities due to “intentional self-harm and assault” on board. In the study by Grappasonni et al. (2012) , 14.3% oThe aim of the present study is to assess the frequency and the sustained shaping of severe stress experiences among seafarers. In addition, it also presents the experiences of shipping companies and Seafarers’ Missions after severe mental stress has occurred on board.An examiner accompanied 22 sea voyages  and interviewed the entire crew about heavy mental loads or potentially traumatic events. Of the 365 crew members, 323 seafarers took part in this study (participation rate 88.5%). Participation in the study was completely voluntary and the data collection was pseudonymised. All participants gave their written informed consent before taking part in this study. The study was approved by the Ethics Committee of the Hamburg Medical Association (no PV4395).The study sample consisted of 156 Europeans (48.3%) and 167 non-Europeans (51.7%). The average age of the exclusively male crew members was 38.2 years (± 11.7 years). For professional stratification, a further distinction was made between the 201 ratings (62.2%) and the 122 officers (37.8%).The recording of the experienced extreme stress and possible psychological after-effects for the crew members after a potentially traumatic event was carried out through a standardized interview. The latter examination procedure based on previously conducted qualitative interviews , 8 as weIn addition, interviews were carried out at 12 German shipping companies: with the owners of the companies (5 times), the managing directors (4 times) or the fleet/crewing managers (2 or 1 time/s). The randomly selected 12 German shipping companies  owned between 1 and 72 container ships and were considered as representative for the German container ship fleet in terms of crew structure and underlying safety standards.Furthermore, a psychologist conducted interviews with the leaders  in 8 stations of the German Seafarers’ Mission.The surveys of the shipping companies and Seafarers’ Missions were based on guided interviews (semi-standardized interviews). A flexible set of questions was compiled during the development of the interview guideline relying on the results of a self-imposed survey of seafarers . The evap-values were two-sided, and a p-value of < 0.05 was considered as statistically significant.Data analysis was performed with SPSS for Windows . Continuous variables were presented as mean (± standard deviation (SD). The Pearson Chi-square test was applied to compare frequencies between groups. All indicated In order to estimate the number of deaths in the study sample, the fatal events in relation to the sum of the seafarer years were calculated in the three-year surveyed period and converted to 100,000 seafarer years .p = 0.003) to often unintentionally think about the shipwreck or the serious accident on board  had experienced ship wrecks or serious accidents. As consequences (according to ICD-10 V (F43.1)), 38.8% of these crew members stated often having to unintentionally think about the smells, 29.3% expressed that they were reminded of the event through certain sounds or smells and 15.5% that they dreamt about it. Officers were significantly more likely than ratings (Fifty-five seafarers (17.0%) experienced a serious robbery or piracy with the after effects of often unintentionally thinking about it (29.1%), being reminded of it through certain sounds or scents (38.2%) and dreaming of the event (9.1%).p = 0.05). In one case, 28 stowaways were discovered in a container and difficulties were encountered in handing them over to the port authorities. After adjusting for age, all observed associations of the cultural and occupational characteristics to the consequences of the traumatic events remained significant.One hundred twenty-six seafarers (39.0%) had experiences with stowaways, more frequently officers than ratings , internal conflicts in treating the stowaways with human dignity, on the one hand, and, on the other hand, having to comply with legal requirements (40.0%), threats to crew (38.1%), high administrative requirements (36.5%), uncertainty about how to behave (34.9%) and physical violence by the stowaways (25.4%).p = 0.021) and ratings  had experienced a threat to the crew by stowaways. This was especially the case for deck crews. Physical violence had tended to be more common among non-European seafarers (and thus many ratings) than among European crew members .In particular, non-European seafarers  was recorded. The 14 deaths represent a crude mortality rate of 78.4 per 100,000 seafarer years.In cases of strong traumatic experiences on board, 3 shipping companies would directly contact the Seafarers’ Mission for initial psychological support. Especially within a Filipino crew, the death of a crew member requires first and foremost pastoral support.If necessary after traumatic experiences on board, some shipping companies would contact the agent (twice), the insurer (twice), the telemedical assistant service (twice) or the maritime rescue centre (twice) for mental health support and also try to replace affected crew members. By hiring out employees, one shipping company made it possible for a smaller shipping company to soon replace a traumatized seafarer.Three shipping companies saw no need for psychological support after extreme stress on board and assessed that this help is the responsibility of the crewing agency alone.According to the interviews with the Seafarers’ Missions, these organizations are particularly active in emergency counselling in the form of crisis intervention. A focal point in the case of a death on board is religious assistance and devotion. After traumatic experiences, the strongly religiously bound Filipino seafarers would try to get in touch with a Catholic priest. Through on-board visits, if possible with longer stay  one-on-one discussions, group discussions, but also devotions were carried out. In cases where no direct personal help in the form of religious assistance or other help is available on the spot, initial contact takes place for about half an hour via internet and skype. A communication of information for a personal debriefing will then be carried out by a native speaker in the ship’s next port of call. A shipping company interested in pastoral-psychological support can facilitate board access by seeing to the necessary formalities, such as visas in advance. The staff of the Seafarers’ Missions is trained for dealing with the tasks of crisis intervention in the English language, which is common in seafaring.The present study revealed that uncontrolled and unintentional thoughts after shipwrecks and personal accidents occurred significantly more frequently among European crew members and especially among officers. Possibly, the officers were more mentally involved in these events because of their leadership responsibility.The Filipino crew with their socio-centered bonds and religious-magical values are likely to be particularly troubled if one of their group members dies or goes overboard and is severely injured. In such cases, feelings of “supernatural” threats arise among the affected Filipino and often result in excessive tendencies to want to escape from the scene of the accident , 13. On Considering potential traumatic events, Maercker and Mehr  distinguAccording to Terheggen et al. , there aAccording to Grover and Ghosh  and BagaBoth physical and mental disorders must also be seen in connection with the socio-economic situation and the family relationship. Being helpless because of mental stress reactions and disorders means a loss of status and face in the family , 25. In Currently, extreme stress is also possible during the rescue of refugees on the high seas. Due to escalating political unrest, the number of refugees fleeing across the Mediterranean has increased in recent years. In the past, merchant ships have been repeatedly involved in large-scale rescue operations of refugees stranded on the high seas. Often the ship’s crews are overwhelmed because of their low personnel levels and, above all, their lack of experience with large-scale rescue operations on the high seas . The somTrauma pilots from the Berufsgenossenschaft Verkehr provide information about possible competent support, treatment options and suitable therapists after a potentially traumatic event on board. Basically, initial psychosocial help after a potentially traumatic event should be given by a neutral, trained person with experience in seafaring who is not involved in the hierarchical structure of a ship’s crew. As, in the closed social system of a ship with a small crew, practically every crew member is indirectly affected by a potentially traumatic event, initial psychosocial help by a suitably trained peer from the crew is not recommended. But this is not a realistic scenario, anyway. For Filipino crews with their strong religious ties, pastoral support should be sought through the Seafarers’ Mission.As far as possible, however, every seafarer, first and foremost the superiors, should have a basic knowledge of the very serious psychological stress caused by shipwrecks, accidents, external violence, etc. Psychoeducation needs to address possible physical and psychological reactions to a potentially traumatic event. It should be noted that the physical and psychological symptoms are normal responses to such an event. This perception can be a relief to the affected crew members. Simple codes of conduct combined with opportunities for short-term relaxation can be very helpful and provide appropriate guidance and support. It is important for anyone affected to gradually find their way back into the structure of everyday life.Psychoeducation for ratings should convey information in simple and understandable words - possibly in the native language - by using appropriate media, such as DVDs. This information should also take cultural perception and experience of extreme mental stress, especially among Asian crew members, into consideration. For European crew members, psychosocial first aid seems to be in the to the experiences of the Seafarers’ Mission, Western Europeans are more planning-oriented and prefer to plan in advance. Asians, on the other hand, “let things happen and only then decide how to react”. This corresponds to Frey and Jonas’ concept of cognized control , i.e. inLeaders should be empowered to recognize signs of psychological overburdening, instability, and shock in their crew members and to provide assistance wherever possible. Psychoeducation should therefore be established as a preventive measure for extreme mental stress situations. Nevertheless, it is not possible to prepare for all crisis situations that can lead to extreme psychological stress. However, it is important to attend to immediate stress reactions and to maintain the control belief. Ideally, according to Davies  and Meic


We analysed typical mammographic density (MD) distributions of healthy Taiwanese women to augment existing knowledge, clarify cancer risks, and focus public health efforts. From January 2011 to December 2015, 88,193 digital mammograms were obtained from 69,330 healthy Taiwanese women . MD measurements included dense volume (DV) and volumetric density percentage (VPD) and were quantified by fully automated volumetric density estimation and Box-Cox normalization. Prediction of the declining MD trend was estimated using curve fitting and a rational model. Normalized DV and VPD Lowess curves demonstrated similar but non-identical distributions. In high-density grade participants, the VPD increased from 12.45% in the 35–39-year group to 13.29% in the 65–69-year group but only from 5.21% to 8.47% in low-density participants. Regarding the decreased cumulative VPD percentage, the mean MD declined from 12.79% to 19.31% in the 45–50-year group versus the 50–55-year group. The large MD decrease in the fifth decade in this present study was similar to previous observations of Western women. Obtaining an MD distribution model with age improves the understanding of breast density trends and age variations and provides a reference for future studies on associations between MD and cancer risk. It is natural and accepted that breast density decreases with increasing age because of postmenopausal glandular breast tissue alterations3. The roles of MD are also included in estimations of adjuvant hormone therapy effects after surgery such as tamoxifen4 and the aromatase inhibitors5. Regardless of associations among MD, breast cancer risk, and endocrine therapy responses, it is important to have a realistic density model to improve accuracy in these related studies.Several studies identified mammographic breast density (MD) as an independent risk factor for breast cancer7; it is interesting to evaluate different breast density distribution patterns between Asian and Western women. Prior Asian population-based studies well reported the association between dense mammograms and age10; however, assessments of longitudinal changes in breast density have used the area-based method11 to measure the two-dimensional area of dense breast tissue on digitized mammography based principally on a semi-automated procedure12. This operator-oriented analysis procedure results in unavoidable bias in dense region selection and limits the number of mammograms that can be studied.Prior studies of breast density among diverse age groups were based mainly on Western women14 and volumetric density measurements from FFDM also provide precise estimates of longitudinal changes in breast density16. It provides an objective and transparent method for density measurements and decreases disagreements among radiologists; this method can also handle large numbers of mammograms and would be useful in providing important prognostic information clinically17. In prior MD distribution studies based on Asian populations, the sample sizes of the cohorts usually ranged from 1,000 to 2,000 and the subjects’ ages ranged from 40 to 65 years. This inspired us to perform an MD distribution analysis based on a large number of FFDM examinations from an Asian population dataset  with all cohorts were from a single country (Taiwan). This can provide high-resolution MD analysis for each age range. We aimed to examine relationships and variations between age and breast density in healthy Taiwanese women by utilizing volumetric density measurements from FFDM with the goals of improving age-specific and risk-specific breast cancer screening guidelines.Recently, full-field digital mammography (FFDM) has advanced the development of automated software that measures volumetric breast density in three dimensionsAfter applying the exclusion criteria, there were 69,330 healthy Taiwanese women who underwent mammography screening who did not have an overlapping record in the breast cancer registry from 2011 to 2015. These women were enrolled and data from 88,193 FFDM examinations were analysed. There were 15,269 participants who contributed multiple FFDM examinations during the study period .Table In this dataset, the median age at the time of mammography was 53 years, the median DV was 44.65  and the median VPD was 12.3 . In the group aged 30 to 49 years, the “d” density grade represented 59.06% of the participants. The density grade “c” represented 51.76% of the participants aged 50 to 69 years. The proportion of participants with a density of “a” or “b” only represented 17.9% of all participants. Table Figure Regarding the naturally decreasing trend in MD, the Lowess curves of the mean normalized VPD and DV Fig.  show thaTable 19; this is a reasonable assumption and was used as a reference in this study.Regarding the naturally decreasing MD in healthy Taiwanese women, the DV/VPD Lowess curve in Fig. 11. By comparing the Lowess curve with the fitted model curve, the association between MD and age in the present study was also similar to that of a meta-analysis combining cross-sectional MD data from 22 countries worldwide12 and a recent Australian population-based study1. This means that the previous conclusion also benefits Taiwanese women by having a similar distribution and decreasing MD trend with increasing age. However, Table The large decrease in MD in the fifth decade of life in the present study was similar to previous observations in Western womenIn the present study, the typical MD distribution in healthy Taiwanese women was shown, as were the relationship and declining trend between MD and age. The results of this study improve the understanding of MD trends and variations with age in Taiwanese women and provide a reference for future related studies on associations between MD and cancer risk. Important limitations to the present study include a lack of information such as the BMIs and menopause statuses of the participants; therefore, the effects of menopause and BMI on MD were not estimated or modelled. This is future work that may result in more information to distinguish different MD distributions in the Taiwanese population than those observed in the present study. We hope that this study will provide sufficient information about normal MD distributions in Taiwanese women.This study was based only on raw FFDM data enrolled from an outpatient clinic and mammography screenings during the period from January 2011 to December 2015; 102,094 FFDM examinations from 77,427 women stored at the institute were subsequently analysed. The ages of the participants ranged from 16 to 95 years. All participants underwent the same procedure and all FFDM examinations were performed at the same institute. No further questionnaire or other data were added to assist in obtaining information such as the body mass index (BMI), menopausal status, or family history.The exclusion criteria were participants with breast cancer, those who underwent a mastectomy on either side, FFDM examinations that lacked the information required for the analysis ; and non-four-view mammograms. Participants were permitted to contribute multiple mammograms because this dataset also includes national mammography screening and follow-up data . Each mammogram was compared with the records of the breast cancer registry at the institute to exclude participants with a breast cancer history and to ensure that participants met the inclusion criteria . Detailed exclusion criteria and the workflow for FFDM data pre-processing are shown in Fig. This cross-sectional retrospective study was approved by the Institutional Review Board of Changhua Christian Hospital, Changhua, Taiwan (No. 160814 and No. 171217). The requirement for informed consent was waived by the ethics committee because of the study’s retrospective nature. All experiments were performed in accordance with relevant guidelines and regulations.All mammograms were obtained from the Department of Medical Imaging at Changhua Christian Hospital, Changhua, Taiwan. All standard two-dimensional digital mammograms used in this study were obtained using FFDM systems including Senographe Essential/Senographe DS  , Mammomat Inspiration  , and Selenia Dimensions  . Each FFDM examination was confirmed to be a bilateral four-view examination before analysis.TM software version 1.5.1 . Briefly, through a specific algorithm that models X-ray physics, the total breast volume (in cm3), the overall dense breast volume (DV) (in cm3) and volumetric density percentage (VPD) estimates were produced20. The VPD was obtained from the DV divided by the total breast volume and multiplied by 100. The DV and VPD in the present study were calculated by averaging the left and right sides.The automated measurement of MD and MD grading were performed using Volpara21. The VDG has already been proven to have a good correlation with radiologist-assigned BI-RADS density categories22; therefore, we did not measure the variance between the VDG and the agreement of the radiologists or combined radiologist-assigned BI-RADS score data.MD grades were reported using the Volpara Density Grade (VDG). The VDG classification is based on quantitative volumetric measurements of fibroglandular and fatty tissue to calculate the VPD  and has different thresholds from the Breast Imaging-Reporting and Data System (BI-RADS) fifth edition23 was used to identify and transform the MD measurements by finding an optimal lambda value  so that their residuals were approximately normally distributed. Locally weighted scatterplot smoothing (Lowess) curves were used to fit the normalized DV and VPD to age (bandwidth = 0.5). It was plotted on the basis of the smoothed VD/VPD and age by applying the greatest weight to the point of interest and decreasing weights for points further away based on their x-axis distance to the point of interest in the dataset24. The independent t test was used to test for differences between each age group and p-values less than 0.01 were considered statistically significant.The MD measurements were transformed to meet the assumption of a mixed-effects model in which the residuals have an approximately normal distribution. The Box-Cox transform method2 value. Rational models with a numerator of degree two and denominator of degree four (rat24) had the best fits after comparisons with several regression models such as linear and cubic/polynomial models and thus were chosen to describe the curve . The formula for the rational models is described in equation  was added to the curve-fitting analysis to obtain a more suitable curve to present the MD distribution of younger women (younger than 40 years). The curve-fitting performance is evaluated according to the model with the minimum residual and highest RAll statistical analyses and regression model analyses were performed using Minitab 18 statistical software (2018) . The curve-fitting for rational models was performed using MATLAB and the Statistics Toolbox Release 2017b .