No clear monogenic links between left-handedness and situs inversus

Left-handedness is a complex trait which might sometimes involve rare, monogenic contributions. Situs inversus (SI) of the visceral organs can occur with Primary Ciliary Dyskinesia (PCD), due to mutations which affect left-right axis formation. Roughly 10% of people with SI and PCD are left-handed, similar to the general population. However, in non-PCD SI, the rate of left-handedness may be elevated. We sequenced the genomes of nine non-PCD SI people who show an elevated rate of left-handedness (five out of nine). We also sequenced six SI people with PCD as positive controls, and fifteen unaffected people as technical controls. Recessive mutations in known PCD genes were found in all positive controls with PCD. Of the nine non-PCD SI cases, two had recessive mutations in known PCD genes, suggesting reduced penetrance for PCD, and one had a recessive mutation in the non-PCD laterality gene PKD1L1. However, six of the nine non-PCD SI cases, including most of the left-handers, had no mutations in likely candidate genes, nor any significant biological pathways affected by their mutations. Therefore we did not identify a molecular link between visceral and brain laterality. While we cannot exclude a monogenic basis for non-PCD SI with left-handedness, multifactorial and non-genetic models must also be considered.


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A fundamental question in human neurobiology is how the brain becomes functionally left-22 right asymmetrical. For example, approximately 90% of people are right-handed and have left- 23 hemisphere dominance for language, among other lateralized functions, but the developmental 24 basis for this asymmetry remains unknown 1 . One possibility is that early embryonic 25 mechanisms which give rise to asymmetries of the visceral organs also impact on brain 26 asymmetries. However, this has not been previously addressed by genetic mutation screening 27 in people who are both left-handed and have altered forms of visceral laterality. 28 Roughly 1:6,000-8,000 people have situs inversus (SI), a mirror reversal of the normal 29 asymmetrical arrangement of the viscera 2,3 . SI can occur alone or in combination with Primary 30 Ciliary Dyskinesia (PCD), a recessive genetic disorder which involves mutations that disrupt 31 motile cilia 2 . Cilia are hair-like organelles that protrude from the cell surface into the 32 extracellular space 4 . They are expressed in various tissues including the respiratory epithelium 33 5 , so that a disruption of ciliary motility can cause symptoms such as chronic bronchitis, 34 inflamed or infected sinuses 6 , which are often present in PCD. 35 Motile cilia are also expressed early in development, within an embryonic structure called the 36 'node', where they generate a leftward fluid flow that helps to create the left-right body axis 5 . 37 The leftward direction of the nodal flow may be explained by a posterior tilt of the cilia together 38 with their clockwise rotation, arising ultimately from molecular chirality of their component 39 proteins 7-9 . When leftward nodal flow is absent due to recessive PCD-causing mutations, many by which these non-PCD, SI-causing genes influence visceral laterality are not well understood, 48 but most code for cilia-associated proteins, rather than coding directly for cytoskeletal 49 components of cilia. 50 Intriguingly, the general population rate of 85-90% right-handedness is not altered in SI people 51 with PCD 16,17 . This implies a developmental dissociation between brain laterality for hand 52 motor control and nodal-ciliary visceral patterning. However, in the only study of handedness 53 in SI to include non-PCD cases, Vingerhoets et al. reported that five of nine SI cases without 54 PCD were left-handed 18 . Although based on a small sample, this suggests developmental 55 mechanisms which might indeed link handedness and visceral laterality, but independently of 56 genes involved in PCD. 57 One study reported a possible genetic link between a continuous measure of left-versus-right 58 hand motor skill and genes involved in visceral laterality 19 , based on analyzing genetic variants 59 which are common in the population. However, the sample size of under 3000 subjects was low 60 for complex-trait genome-wide association analysis using common genetic variants. A much 61 larger study of over 300,000 subjects from the UK Biobank found no support for a link of left-62 handedness to genes involved in visceral asymmetry 20 . The same large study identified an 63 association of a common variant at the MAP2 gene with left-handedness, with a very small 64 effect 20 . Left-handedness has a heritability of roughly 25% based on twin and family data 21 , 65 but only around 2% based on genome-wide genotype data for common polymorphisms, within 66 the UK Biobank dataset 22 . 67 It has been proposed that left-handedness may sometimes occur due to genetic mutations which 68 are rare in the population, but might have substantial effects on brain laterality when present  group. In addition, all six members of the PCD group reported having a daily wet cough, and 99 had PICADAR scores of between 8 and 12 25 , and thus predictive probabilities of having PCD 100 of between 66% and 99%, based on this recently-developed, questionnaire-based tool (note that 101 five of these subjects anyway had formal medical diagnoses).

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A seventh SI subject (SI03) had no medical record pertaining to PCD but did report daily wet 103 cough. This subject had a PICADAR score of 8. In the study of Vingerhoets et al. this SI subject 104 was classified as non-PCD, before the PICADAR assessment was available. For the purposes 105 of the present study, we also treated this subject as non-PCD given the lack of formal medical 106 history of PCD and the ambiguous PICADAR score, but we repeated some genetic analyses 107 having excluded the person from the non-PCD group, in order to account for this uncertainty 108 (see below).

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Eight other SI subjects had no medical record of PCD or PCD-like symptoms, and were 110 classified as non-PCD SI cases. Six of these reported no daily wet cough, and two did not 111 answer in this regard. PICADAR scores can only be calculated in the presence of daily wet 112 cough, so that none of these eight cases received PICADAR scores. Three of these cases had 113 been previously diagnosed with congenital heart disease that required surgical treatment, and 114 their radiological files all referred to their cardiac condition. Congenital heart disease is a 115 frequent comorbidity of SI, as the cardiac circulation appears particularly sensitive to 116 perturbation in normal left-right positional information 26 .

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Handedness was assessed using the Edinburgh Handedness Inventory (EHI) 27 . Note that one 118 non-PCD SI subject reported being forced to switch from left-to right-handedness in childhood, 119 in which case five out of nine non-PCD SI cases were naturally left-handed. One of the six 120 cases with PCD also reported forced left-to-right switching, otherwise the rest were right-121 handed ( Table 1).

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Whole Genome Sequencing (WGS) and Pre-processing 123 DNA was extracted from saliva samples using the Oragene kit (Oragene). WGS was performed 124 by Novogene (Hong Kong) using Illumina high throughput sequencing (HiSeq-PE150), 125 creating paired end reads with a length of 150 base pairs (bp). Raw reads, stored in BAM files, 126 were aligned to the human reference genome (the extended "decoy" version of b37) using 127 Burrows-Wheeler Aligner (BWA) software 28 , and sorted and reordered using SAMtools 128 (v1.3.1) 29 . PCR duplicates, which could arise during cluster amplification, were marked using 129 Picard (v2.9.0). Genome Analysis Toolkit (GATK v3.7) 30 software was used to realign reads 130 around insertions/deletions (indels) and to recalibrate base quality scores per sample. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.   CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/422964 doi: bioRxiv preprint first posted online Sep. 21, 2018; A maximum population frequency threshold of 5×10 -5 was applied in this case, and genes 185 carrying at least one rare variant according to this criterion were considered as potentially 186 causative. Again, genes mutated according to this criterion in any of the fifteen unaffected 187 control subjects (N = 47 genes) were excluded as being potentially causative in cases, and 188 removed for the purposes of GSEA analysis (below).

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To test whether a list of mutated genes in a given set of subjects contained functionally related 191 genes across subjects, we performed Gene Set Enrichment Analysis (GSEA) using the   The identities of genes were first converted using the g:Convert tool 41 to ensure recognition by 220 the GO schema. The following settings were then used for GSEA: minimum set size = 15, 221 maximum set size = 500, minimum intersect number = 2, hierarchical filtering = moderate. P-222 values were corrected for multiple testing across gene sets, based on the gSCS correction 223 method in g:Profiler, separately for each input list of mutated genes corresponding to a given 224 set of subjects. This method of multiple testing correction takes into account the hierarchical 225 structure of the sets 41 . We applied a cut-off P value of adjusted 0.01.

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Candidate Gene Lists 227 We queried the genetic data with respect to candidate gene lists for some purposes (see Results).

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An initial list of candidate genes was created in R (v3.3), based on searching the Online 229 Mendelian Inheritance in Man (OMIM) database 44 for the terms: "situs inversus", "heterotaxy 230 | heterotaxia | situs ambiguus", "congenital heart disease", "PCD | ciliary dyskinesia | 231 . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/422964 doi: bioRxiv preprint first posted online Sep. 21, 2018; Kartagener", "left-right", and "asymmetry | laterality". Additionally, the Clinvar column of our 232 annotated variant data, which contains information based on the Clinvar database 45 , was 233 searched for these terms, and genes that were not yet in the initial list of candidate genes were 234 accordingly included.

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A broader candidate gene list was also created which included genes from the literature that 236 were suggested to be associated with human laterality phenotypes and/or ciliopathies, but not 237 (yet) present in our initial list of candidate genes (Supplementary Table S1). Specifically, a 238 list of ciliopathy genes from a review of this topic 6 was searched for the terms: "PCD", 239 "heterotaxy", "situs", "left-right", "asymmetry" or "laterality", yielding 18 additional candidate where it was 150 base pairs (we determined a roughly optimal bin size for each subject's 256 genome, such that that the average read depth for that genome would be at least 4 standard 257 deviations from zero). Lumpy was run using default parameters in "lumpyexpress".

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SVs were then annotated using SV2 53 . Variants that were present in any of the 15 healthy 259 controls were removed from consideration as potentially causative for SI. Only variants that 260 passed the default SV2 filtering criteria for quality were included 53 .  Table 1). The PCD subject with a homozygous LRRC6 mutation (subject SI06) 277 . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/422964 doi: bioRxiv preprint first posted online Sep. 21, 2018; was the only individual to show an elevated inbreeding coefficient and non-European ancestry 278 (Supplementary Table S2, Supplementary Figure S1). 279 None of the fifteen unaffected control subjects had any recessively mutated genes annotated 280 'Kartagener', 'PCD', 'SA' or 'SI' in Clinvar. 281 Surprisingly, two of the nine non-PCD SI cases had recessive mutations in genes annotated 282 'Kartagener' in the Clinvar database 45 . These were subject SI12 (again involving mutations in 283 DNAH5), and subject SI16 (in CCDC151) ( Table 1). As these subjects had no medical records 284 pertaining to PCD-like symptoms, and no daily wet cough, then the findings suggest reduced 285 penetrance for PCD.

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One of the nine non-PCD subjectsi.e., SI02 -had a recessive mutation in a gene that is 287 annotated in Clinvar as 'situs ambiguus' and 'situs inversus totalis', but not annotated as PCD-288 causing ( Table 1) (Table 1). Among these six non-PCD 299 SI cases, four were left-handed, and were therefore of primary interest for the present study.

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Three of these same subjects also had CHD (Table 1). 301 We constructed an extended list of known or suspected laterality genes with reference to the 302 literature and mouse laterality phenotypes (Methods; Supplementary Table S1), but none of 303 these genes had recessive mutations in the six unsolved non-PCD SIT subjects.  and since there was no such diagnostic record for this subject, one or both of these specific 308 mutations probably has limited functional impact and is therefore unlikely to be a monogenic 309 cause for SI either.  CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.  Table 3), which was included among our candidate genes. However, since recessivebut not 328 dominant -mutations in this gene have been associated with PCD 61 , it is unlikely that this 329 mutation is causal for non-PCD SI in this subject. 330 We also note a heterozygous mutation in WDR62 [MIM:613583] in the unsolved case SI09  Gene set enrichment analysis 350 We first performed gene set enrichment analysis using the positive control set of six SI subjects 351 with PCD. As noted above, the six PCD subjects had likely recessive monogenic causes in five 352 different genes (two subjects had mutations in the same gene, DNAH5). As expected, from the 353 total combined list of recessively or X-linked mutated genes in these subjects, of which 40 354 genes were included in the GO schema, we obtained significant results for various cilia-related  With this in mind, we then performed gene set enrichment analysis in the non-PCD SI cases, 367 who were of primary interest for the present study due to a potential link with left-handedness. 368 However, no significant enrichments were found when testing the list of recessively or X-linked 369 mutated genes in the nine subjects with non-PCD SI, of which 38 genes were included in the 370 . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint . http://dx.doi.org/10.1101/422964 doi: bioRxiv preprint first posted online Sep. 21, 2018; GO schema. There was also no significant functional enrichment when testing the recessively 371 mutated or X-linked genes in the five left-handed subjects with non-PCD SI, of which 26 genes 372 were in the GO schema, and neither when testing the list of genes in the six unsolved non-PCD 373 SI subjects, of which 22 genes were present in the GO schema ( Table 2). Repeating the analysis 374 after excluding subject SI03 from these subsets made no difference, there were still no 375 significant gene sets. In addition, gene-set enrichment analysis with dominantly mutated genes 376 as input did not produce significant results in the non-PCD SI cases, nor the left-handed or 377 unsolved subsets.

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As expected, the lists of recessively/X-linked and dominantly mutated genes in the fifteen 379 unaffected control subjects did not produce any significant gene set enrichment terms (Table   380 2). 382 We additionally screened the genomes of all subjects for structural variants (such as larger- In this study we aimed to identify rare, highly penetrant genetic mutations that might link 392 visceral body asymmetry with handedness, by analysing whole genome sequence data from 393 nine SI subjects without medical histories of PCD, five of whom were left-handed. We CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

Structural variant analysis
The copyright holder for this preprint . http://dx.doi.org/10.1101/422964 doi: bioRxiv preprint first posted online Sep. 21, 2018; the subset that was 'unsolved' by recessive monogenic causes. In other words, the biology of 417 their non-PCD SI could not be linked via the mutations that they carried. Finally, we also 418 considered larger genomic rearrangements known as Copy Number Variants (CNVs), but no 419 obvious candidate genes emerged. 420 A monogenic model is still possible for the majority of the non-PCD SI cases, and/or for the 421 left-handed subset specifically, but would have to involve genetic heterogeneity across a set of 422 genes which are not currently linked in terms of their known biology, at least to an extent which 423 would have been discernible in this dataset, as it was for the PCD subjects. We did not therefore 424 identify a genetic-developmental pathway that links handedness and visceral asymmetry in this 425 study. The question of whether, and how, functional brain laterality is linked developmentally 426 to visceral laterality in humans remains unanswered 20 .

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Genetic contributions to non-PCD SI and left-handedness might also involve non-coding 428 variation, or rare combinations of multiple common variants. The noncoding genome comprises 429 98% of the genome, but interpreting the variation within these regions is challenging. Several 1 Francks, C. Exploring human brain lateralization with molecular genetics and genomics. . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. . CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.