FMR1 allele size distribution in 35,000 males and females: a comparison of developmental delay and general population cohorts
Short title: FMR1 allele size distribution in 35,000 males and females
Claudine M. Kraan, PhD1,2,3, Quang M. Bui, PhD4, Mike Field, MBChB, MPhil5, Alison D Archibald, PhD1, Sylvia A Metcalfe, PhD2,6 Louise M Christie5, Bruce H Bennetts, PhD7, Ralph Oertel, PhD1, Melanie J Smith1, Desiree du Sart, PhD, FFSc(RCPA)1, Damien Bruno, PhD, FFSc(RCPA)1, Tiffany L Wotton, PhD7,8, David J Amor, MBBS, PhD1,2, David Francis, MSc, FFSc (RCPA)11 & David E Godler, PhD1.
Supplementary Note S1: Extended sample characteristics
Supplementary Table S1. PM and GZ results in developmentally delayed cohorts
Supplementary Table S2: PM and GZ results in females: pairwise comparison (corresponds to Table 2)
Supplementary Table S3. PM and GZ results, including positive FMR1-family history data
Supplementary Table S4: PM and GZ results in females, including positive FMR1-family history data: pairwise comparison (corresponds to Table S3)
Supplementary Table S5. PM and GZ results in DD cohorts, inclusive and exclusive of positive FMRI-family history data
Supplementary Table S6. Estimated Australian population prevalence rates in DD and population cohorts, stratified FMR1-family history
Supplementary Table S7. Frequency of ‘low normal’ allele results in Australian proband DD and population screening cohorts
Supplementary Table S8: Female homozygous ‘low normal’ allele results: pairwise comparison (corresponds to Table S7)
Supplementary Figure S1. Female CGG size distribution plots for the larger FMR1 allele.
Supplementary Note S1: Extended sample characteristics. TheDD #1 cohort included children ≤18 years old referred by a clinician to VCGS between 2003 and 2009. A larger version of this cohort that included a broader age range of1 week to 89.9 years has previously been published.1All cases with a final result in this cohorthad either clear results on Polymerase chain reaction (PCR) testing or had confirmatory Southern blot testing, either because PCR detected ‘one peak’ in a female or ‘no peak’ in a male. These data do not include specific details of how many cases went for Southern blot testing, nor does it include CGG size. A total of 39 cases withFM alleles were removed from the cohort. An additional 70 cases were found in the cohortthat did not have a final result and thus were deemed inconclusive as they had not been followed up (6 male, 64 female), plus there were a further 5 cases in the cohortthat did not have gender listed (all normal FMR1 genotypes). Only non-FM cases with a confirmed final result and gender were included in the dataset for final analyses (N= 10,235).
The DD#2cohort from VCGS, which was created between 2013 and 2017, was more comprehensive than the DD #1 cohort due to inclusion of CGG size results. In this cohort there were 504 inconclusive PCR results that may have represented either a normal homozygous result (i.e., 2 identical sized alleles) or a single normal allele with an undetected expanded allele. All cases were sent for further Southern blot and/or microarray testing for confirmation. Of these, there were 7 that went to microarray and had a confirmed chromosomal abnormality and 7 that went for Southern blot but the attempted test failed to produce a result or there were insufficient DNA for testing. Additionally, there were 12 cases that did not go to Southern (with reason unclear) and a further 4 ‘two-peak’ males that were not sent for microarray testing and thus remained unclear regarding presence of a chromosomal abnormality. These cases, where the final result was not clear, or there was a chromosomal abnormality (n= 30), were not included in the final cohort. Of the remaining 481 inconclusive cases that had reliable results, most (n=455) had been sent for confirmatory testing due to finding on PCR of a ‘1 peak’ female. A total of 444 of this group were confirmed to be normal by Southern blot. The remaining 11 females with ‘1 peak’ results on PCR were found to be abnormal after Southern blot testing (3 premutation/full mutation (PM/FM), 7FM and 1 PM). Another 5 cases (all male) were determined to be inconclusive based on PCR testing due to presence of a ‘low peak’, with 3 found to be FM and 2 with normal alleles detected with Southern blot. There were a further 20 cases with ‘no peak’, comprising 4 PM/FM males, 6 FM males, and 9 males and 1 female with a normal FMR1 allele. Plus, there was one male with ‘one low peak’ who was determined to have a normal FMR1 allele. All 8841 cases that were confirmed to not have FM or PM/FM alleles (N=23) ora chromosomal abnormality were included in the cohort for analyses.
The adultcarrier screeningcohortwas created between 2013 and 2017, and included diagnostic workflow, CGG size, symptoms consistent with a FXS-associated disorder and knowledge of an FMR1 expansion in a blood relation. There were 42 cases not included in the final cohort, these reflected females who requested to not have FXS tested (n=21), tests that were cancelled with the result not issued (n=3), recollect reports (n=17) and 1 FM female that was picked up. All remaining 14,249 cases were analysed. A total of 59 cases had Southern blot confirmatory testing (only 1 failed). Reasons for confirmatory testing included FXPOI symptoms (1 case, normal result), family history of an FMR1 expansion ticked on the study leaflet (n=21) and no family history but follow up/confirmation of an expanded or unclear FMR1 allele on PCR (n= 38). Positive “grey zone”(GZ) samples were not sent for confirmatory testing with Southern blot. All but one sample with a PM result on PCR were confirmed through Southern blot; and one additional PM case (CGG 23, 56) failed on Southern blot.
The newborn screening cohortincluded one boy with Klinefelter syndrome who was removed before the final analyses were performed. We also removed two children as their gender was unclear. Analyses were performed on 1997 cases.
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Supplementary Table S1. PM and GZ resultsin developmentally delayed cohorts
ReferenceFirst author and year / Method / GZ / PM / CGG distribution
Cohort
Age
Nation / Cohort: Percentage
(N positive/sample size);
Estimated prevalence / Cohort: Percentage
(N positive/sample size);
Estimated prevalence
2Khaniani et al., in press / Cohort: Autism (96 males)
Cohort: Control (132 males)
Age: 2-20 (Autism); 6-18 (control)
Nation: Iran / Autistic males: 5.2% (5/96); 1 in 19
Control males: 0
*GZ defined as 40-54 / Autistic males: 4.2% (4/96); 1 in 24
Control males: 0.76% (1/132); 1 in 132 / Not mentioned
3Kanwal et al., 2015 / Cohort: Intellectual disability (ID) (395 cases: 287 males; 108 females)
Age: 4 to 40 years
Nation: Pakistan / 0 / 0 / Not mentioned
4Viveiros et al 2015 / Cohort: ID (238 males)
Age: 4 to 60 years
Nation: Brazil / 0 / 0 / Not mentioned
5Chen et al., 2015 / Cohort: ID (540 cases: male:female = 5.2:1)
Age: 6 months to 18 years old
Nation: China / 0 / 0 / Mode: 29 and 30
6Fatima et al., 2014 / Cohort: ID (333 cases)
Cohort: Typically developing (TD) (250 cases)
Age: 5 to 18 years
Nation: Pakistan / 0 / 0 / ID: Mode: 29; second peak 28; third peak: 30
7Tassone et al., 2013 / Cohort: TD (346 cases: 262 males; 84 females)
Cohort: Autism (309 cases: 270 males; 39 females)
Cohort: Autism Spectrum Disorder (ASD) (144 cases: 121 males; 23 females)
Cohort: Developmental delay (DD) (146 cases: 95 males; 51 females)
Age: 15- 72 months
Nation: US population / TD males: 0.4 % (1/262); 1 in 262
Autism/ASD males: 1.3 % (5/391); 1 in 78
DD males: 0
TD females: 2.4 % (2/84); 1 in 42
Autism/ASD females: 3.2 % (2/62); 1 in 31
DD females: 1.96% (1/51); 1 in 51 / TD males: 0
Autism/ASD males: 0
DD males: 1% (1/95); 1 in 95
TD females: 0
Autism/ASD females: 0
DD females: 1.96% (1/51); 1 in 51 / Not mentioned
8Essop et al., 2013 / Cohort: Genetic testing referrals for ID (2239 cases: males and females)
Age: Not mentioned
Nation: South African / Genetic testing referrals: 1.8% (41/2239); 1 in 54
*Of the 41 GZ’s, 31 were male and 10 were female / Genetic testing referrals: 0.54% (12/2239); 1 in 187
*Of the 12 PM’s, 8 were male & 4 were female / ID: Mode: 29; second peak: 30
*distribution described as different between black and white populations
9Renda et al., 2012 / Cohort: Clinical group (1447 cases; male/female proportion not specified)
Age: birth to 18 years old
Nation: US population
*cannot analyses males and females separately because of lack of male/female proportion in total group examined
*for above reason cannot calculate prevalence in cohort / Clinical group: 1.94% (28/1447); 1 in 52
*Important to note that this study ruled out other genetic or environmental causes
*of those that consented to review of medical records all have at least 1 neurodevelopmental diagnosis / Clinical group: 0.35% (5/1447); 1 in 289
*Important to note that this study ruled out other genetic or environmental cause
*of those that consented to review of medical records all have at least 1 neurodevelopmental diagnosis / Not mentioned
10Madrigal et al., 2011 / Cohort: TD (5775 males)
Cohort: ID (9015 males)
Cohort: Attention Deficit Hyperactivity Disorder (ADHD) (415 males)
Cohort: ASD (300 males)
Age: 18 months to 45 years old
Nation: Spain
*diagnosis based on DSM-IV
Criteria / TD males: 3.5% (204/5775); 1 in 28
ID males: 1.6% (142/9015);
1 in 64
ADHD Males: 0.98% (4/415);
1 in 104
Males with ASD: 1.33% (4/300); 1 in 75 / 0 / NA
11Otsuka et al. 2010 / Cohort: TD (1161 cases: 513 males; 324 females)
Cohort: ASD (116 cases: 102 males; 14 females)
Age: Not mentioned
Nation: Japan / TD males: 0.98% (5/513); 1 in 103
TD females: 0.31% (1/324); 1 in 324
ASD: 0
(Males & females analysed together): Not detected
*GZ defined as 40-54 / 0 / TD: Mode: 27; minor peak: 26; minor peak: 34
12Mitchel et al., 2005 / Cohort: Children in Special Education Needs (SEN) classes (1253 males)
Cohort: Newborns (578 males)
Age: 5 to 18 years old; newborn
Nation: Australia / SEN males: 3.4% (43/1248); 1 in X
Newborn: 2.4% (14/575); 1 in X
*GZ defined as 41-60
* Increased GZ frequency in SEN group versus control / 0 / Both samples: Mode: 30l minor peak: 20; minor peak: 23
13Youings et al., 2000 / Cohort: Children in SEN classes (3738 males)
Cohort: Untransmitted Maternal allele (2968 alleles)
Age: 5-18 years old
Nation: England / SEN males: 3.8%% (141/3732); 1 in 27
Untransmitted: 2.7% (79/2932); 1 in37
*GZ defined as 41-50
*Significant increase of GZ+PM in SEN males compared to control / SEN males: 0.05% (2/3732); 1 in1866
Untransmitted: 0.03% (1/2932); 1 in 2932
*PM defined as 61-200 CGGs
*Significant increase of GZ+PM in SEN males compared to control / Not discussed
14Haddad et al., 1999 / Cohort: ID (251 males)
Cohort: TD (251 males)
Age: School-age
Nation: Brazil
*randomly selected from special schools / ID males: 6.4% (16/251); 1 in 16
Control males: 2.8% (7/251); 1 in 36
*GZ defined as 41-60 / 0 / Not discussed
15Crawford et al., 1999 / Cohort: Children in SEN classes (2851cases: 1979 males; 872 females)
Age: 7-10years old
Nation: US population / SENmales:3.9% (78/1979); 1 in 25
SEN females: 5.5% (48/872); 1 in 18
*GZ defined as 41-60 / SEN males: 0
SEN females: 0.23% (2/872); 1 in 436
*PM defined as 61-199 / SEN: Allele sizes collapsed
into three categories
Gp. 1. 11–26 repeats,
Gp. 2. 27–34 repeats
Gp. 3. >34 repeats
* lower frequency of Gp. 1 and Gp. 3
in the African American compared
with white cohort.
16Mila et al 1997 / Cohort: ID (222 cases: 182 males; 40 females)
Age: 4 to 20 years old
Nation: Spain / 0 / 0 / Not mentioned
17Murray et al., 1996 / Cohort: Learning disability (LD) (1013 males)
Cohort: Maternal X chromosome (726 females)
Age: 5-18 years old
Nation: United Kingdom / LD males: 3.46% (35/1013); 1 in 29
Maternal X Chromosome: 1.93% (14/726); 1 in 52
*GZ defined as 41-60 CGGs
*Excess GZ in LD group compared to maternal X chromosome. / LD males: 0.1% (1/1013); 1 in 1013
Maternal X Chromosome: 0
*PM defined as 61-200 / Combined group: Mode: 30;
minor peak: 20
Note:*Where GZ and PM classifications are different to what is used currently in standard practice (i.e., GZ: 45-54; PM: 55-199) the definition is shown in italics: This table is not exhaustive as itdoes not include meta-analysis studies, studies in older neurological cohorts, studies using very dated molecular diagnostic methodologies and studies that could not be sourced. Reported numbers are based on the number of samples available for CGG sizing.
ASD = Autism Spectrum Disorder; ADHD = Attention-Deficit Hyperactivity Disorder; DD = developmental delay; ID = intellectual disability; LD = learning disorder; SEN = special education needs; TD = typically developing
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Supplementary Table S2: PM and GZ results in females: pairwise comparison (corresponds to Table 2)
p1 / p2 / p3 / p4 / p5 / p6PM / 0.783 / 0.999 / 0.035 / 0.656 / 0.091 / 0.008
GZ / 0.188 / 0.281 / 0.213 / 0.008 / 0.876 / 0.032
Note:Pairwise comparisons corresponding to female data in Table 2:1DD #1 vs DD #2; 2DD #1 vs adult carrier screening; 3DD #1 vs newborn screening; 4DD #2 vs adultcarrier screening; 5DD 2 vs newborn screening; 6adult carrier screening vs Newborn screening.
p = p-value; GZ = “grey zone”; PM = premutation.
p-value in bold was < 0.05 after adjustment for multiple comparison using false discovery rate (FDR).
Supplementary Table S3. PM and GZ results,including positive FMR1-family history data
DD #1(2003-2009)a / DD #2
(2013-2017) / Newborn screening / Adultcarrier screening / p*
n+ (n-) % / n+ (n-) % / n+ (n-) % / n+ (n-) %
Male
PM / 22 (7831) 0.28 / 10 (6663) 0.15 / 2 (1014) 0.20 / - / 0.246
GZ / 55 (7798) 0.70 / 51 (6622) 0.76 / 8 (1008) 0.79 / - / 0.848
Female
PM / 12 (2370) 0.50 / 11 (2157) 0.51 / 8 (973) 0.82 / 44 (14,205) 0.31 / 0.030
GZ / 52 (2330) 2.18 / 35 (2133) 1.61 / 14 (967) 1.43 / 359 (13,890) 2.52 / 0.011
Note: Data in this table include individuals in whom presence of an FMR1 expansion in a blood relation was indicated. n+ (n-): number of positive results (number of negative results); DD #1: paediatric DD referrals to VCGS between January 2003 and December 2009; DD #2: paediatric DD referrals to VCGS between September 2013 and April 2017.
*p-value comparing equality of proportion across the four cohorts was computed using Fisher’s exact test. Significant results were followed up with pairwise analyses in Supplementary Table S4.
Supplementary Table S4: PM and GZ results in females,including positive FMR1-family history data: pairwise comparison (corresponds to Table S3)
p1 / p2 / p3 / p4 / p5 / p6PM / 0.999 / 0.128 / 0.324 / 0.159 / 0.324 / 0.017
GZ / 0.193 / 0.354 / 0.172 / 0.008 / 0.758 / 0.032
Note:Pairwise comparisons corresponding to female data inclusive of individuals in whom presence of an FMR1 expansion in a blood relation was indicated in Supplementary Table S3:1DD #1 vs DD #2; 2DD #1 vs adultcarrier screening; 3DD #1 vs newborn screening; 4DD #2 vs adult carrier screening; 5DD 2 vs newborn screening; 6adultcarrier screening vs newborn screening. p = p-value; GZ = grey zone; PM = premutation.
p-value in bold was 0.05 after adjustment for multiple comparison using false discovery rate (FDR).
Supplementary Table S5. PM and GZ resultsin DDcohorts, inclusive and exclusive of positive FMRI-family history data
Indicatedfamily historyA / No family historyB / p-value*
n+ (n-) % / n+ (n-) %
DD #1 Male PM / 22 (7831) 0.28 / 17 (7814) 0.22 / 0.224
DD #1 Male GZ / 55 (7798) 0.70 / 53 (7778) 0.68 / 0.837
DD #2 Male PM / 10 (6663) 0.15 / 8 (6633) 0.12 / 0.475
DD #2 Male GZ / 51 (6622) 0.76 / 46 (6595) 0.69 / 0.460
DD #1 Female PM / 12 (2370) 0.50 / 6 (2347) 0.26 / 0.023A
DD #1 Female GZ / 52 (2330) 2.18 / 50 (2303) 2.13 / 0.999
DD #2 Female PM / 11 (2157) 0.51 / 7 (2138) 0.33 / 0.131
DD #2 Female GZ / 35 (2133) 1.61 / 34 (2111) 1.59 / 0.863
Note:n+ (n-): number of positive results (number of negative results); DD #1: paediatric DD referrals to VCGS between January 2003 and December 2009; DD #2: paediatric DD referrals to VCGS between September 2013 and April 2017; GZ = “grey zone”; PM = premutation.DD clinical notes and adultcarrier screening test request forms A indicate an FMR1 expansion in a blood relation; Bdo not document knowledge of an FMR1 expansion in a blood relation.
Exact p-value for testing the proportion of group inclusive of family history data is equal the proportion exclusive of family history data, using binomial probability test.Ap-value was > 0.05 after adjustment for multiple comparison using false discovery rate (FDR).
Supplementary Table S6. Estimated Australian population prevalence rates in DD and population cohorts, stratified FMR1-family history
Inclusive of +ve FMR1 family history data / Exclusive of +ve FMR1family history data
Male / Female / Male / Female
PM
DD #1 / 1/357 / 1/199 / 1/461 / 1/392
DD #2 / 1/667 / 1/197 / 1/830 / 1/306
Newborn screening / 1/507a / 1/123a / 1/507a / 1/123a
Adult carrier screening / N/A / 1/324 / N/A / 1/374
GZ
DD #1 / 1/142 / 1/47 / 1/148 / 1/47
DD #2 / 1/131 / 1/62 / 1/144 / 1/63
Newborn screening / 1/127a / 1/70a / 1/127a / 1/70a
Adult carrier screening / N/A / 1/40 / N/A / 1/40
Note: DD #1: paediatric DD referrals to VCGS between January 2003 and December 2009; DD #2: paediatric DD referrals to VCGS between September 2013 and April 2017; GZ = “grey zone”; PM = premutation. aThe newborn screening cohort did not include information on family history of an FMR1 expansion, thus the same result is presented for newborn screening cohort in each column. N/A= no data available.
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Supplementary Table S7. Frequency of ‘low normal’ allele results inAustralian proband DD and population screening cohorts
DD #2 / Newborn screening / Adult carrierscreening / p*
n+ (n-) % / n+ (n-) % / n+ (n-) %
Prevalence / Prevalence / Prevalence
Male / 1265 (5408) 18.96% / 216 (800) 21.26% / - / 0.09
~1 in 5 / ~1 in 5
Female (at least one allele) / 748 (1420) 34.50% / 330 (651) 33.64% / 4997 (9252) 35.10% / 0.61
~1 in 3 / ~1 in 3 / ~1 in 3
Female (homozygous) / 65 (2103) 3.00% / 53 (928) 5.40% / 590 (13,659) 4.14% / 0.004
~1 in 33 / ~1 in 19 / ~1 in 24
Note:Cohorts analysed are inclusive of all data, including individuals in whom presence of an FMR1 expansion in a blood relation was indicated.n+ (n-): number of positive results (number of negative results); DD #1: paediatric DD referrals to VCGS between January 2003 and December 2009; DD #2: paediatric DD referrals to VCGS betweenSeptember 2013 and April 2017. *p-value comparing equality of proportion across the four cohorts was computed using Fisher’s exact test. Significant results were followed up with pairwise analyses (Supplementary Table S8)
Supplementary Table S8: Female homozygous ‘low normal’ allele results: pairwise comparison (corresponds to Table S7)
p1 / p2 / p3Hom. ‘low normal;’ allele / 0.002 / 0.011 / 0.059
Note:Pairwise comparisons corresponding to female data in Supplementary Table S7: 1DD #2 vs NBS; 2DD #2 vs adult carrier screening; 3newborn screening vs adult carrier screening. p-value in bold was < 0.05 after adjustment for multiple comparison using false discovery rate (FDR). Hom = Homozygous for ≤ 26 CGG repeats.
Supplementary Figure S1. Female CGG size distribution plots for the larger FMR1 allele. CGG size distribution plotswith CGG size on the X-axis (0 -70 repeats) and percentage on Y-axis: (A): DD #2 (N= 2168); (B) newborn screening (N= 981); (C) adultcarrier screening (N= 14,239).
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