To the Editor of Journal of Medical Genetics:

Enabled by recent advances in sequencing technologies, genotypes from thousands of individuals are now available in online databases. While most of them aim to be the reference source of genotypes from healthy individuals, however, due to the lack of accompanying clinical data, geneticists now face the challenge of separating pathogenic mutations and rare polymorphisms. The frequency of pathogenic variants within a population database could be inflated by subjects who are not yet diagnosed or misdiagnosed. In particular, it is more challenging for geneticists working on late on-set neurodegenerative diseases, such as SCA40 (1), where pathogenic variants may lurk within the genome for decades, until gradual deterioration of symptoms could be observed. In the case of SCA34, only 63% of ELOVL4 L168F carriers demonstrated ataxia (2). This is possibly due to the relatively young age of carriers (less than 48 years old), and thus the ataxia symptoms take time to develop (2).

The ExAC database (3) is one of the largest genotype databases to date, which combines sequencing data from 60,706 unrelated individuals in cohorts such as National Institute of Mental Health Controls, schizophrenia, bipolar disorder, Tourette's syndrome, and The Cancer Genome Atlas (TCGA). This clearly shows that pathogenic variants can be found in ExAC, and thus it might not be suitable to be treated as the genetic background of healthy population. MacArthur et. al. (3) have clearly stated that ExAC was designed to be the reference data set for childhood-onset Mendelian diseases, and the subjects are free of known severe pediatric diseases only[1]. Therefore, it is questionable to claim a variant as rare polymorphism based on ExAC alone.

To illustrate the prevalence of neurodegenerative diseases markers in population genetics databases, we have screened all known markers of autosomal dominant Spinocerebellar Ataxia (SCA) and Spastic Paraplegia (SPG) other than SCA40 (Table 1). Altogether, 12 SCA markers and 6 SPG markers were found in the analysis with allele frequency ranging from 8.236e-06 to 0.0262. This reaffirms the notion that subjects with neurodegenerative diseases may exist in population genetics databases. It is known that the pathogenicity of a disease marker can change in different ethnicity background (4, 5). Ethnic specific modifiers may alter the penetrance of these markers, accounting for higher frequency of these markers in certain populations (5, 6). Since SCA40 marker induces apoptosis through JNK pathway activation (1), the relative activity of JNK1/2/3 and c-Jun could modulate the levels of apoptosis (7-9), regulating the penetrance of SCA40 marker as a result.

Besides, we believe the eLetter authors misinterpreted 1000 genome phase III frequency for CCDC88C R464H, since only R464C and R464S (rs371123543) could be found in the data according to NCBI 1000 Genome browser (Figure 1). To date, CCDC88C R464H is still unreported in 1000 Genomes phase III, 1KJPN (10), ESP6500, GoNL (11), SGVP (12), and UK10K ALSPAC/TWINSUK cohort (13). The absence of CCDC88C R464H in 1KJPN database, which curates the whole-genome sequences of 1,070 healthy Japanese individuals, clearly contradicts with the eLetter authors' claim that the variant is relatively common in Japanese control alleles. In our original study, we screened 199 local healthy subjects, but none of them harbours R464H. The eLetter authors also independently screened 24 local healthy subjects and 85 disease controls, yet the variant was only found in one patient with SOD1-associated autosomal dominant amyotrophic lateral sclerosis (ALS). Given the extremely low frequency of CCDC88C R464H variant in the population, it is unlikely that R464H represents a rare polymorphism. Of note, cerebellar ataxia was previously reported in an ALS case associated with SOD1 variants (14), so the cerebellar features of the patient containing both SOD1 and CCDC88C R464H variants merits further investigation.

To explore the possibility of finding additional disease markers in our patient samples after the publication of our study in 2014, we now reanalyzed the sequencing data using the latest human genome reference (GRCh38), updated BWA alignment tool (version 0.7.15) (15), GATK haplotypecaller variant caller (Version 3.5) (16), Clinvar database (build 20160831), dbSNP 147, and 1000 genome database phase 3, yet no other known pathogenic marker was further identified. In addition to our in-house variant prioritization method as outlined in our original study, we did not observe any discrepancy after cross-checking our results against KGGSeq results (17). Together with the support of multipoint parametric genetic linkage analysis, gene expression profile mining and functional impact predictions as described in our study, we believe we have gathered substantial in silico evidence to support CCDC88C R464H as a disease marker.

In addition to the in silico predictions that the CCDC88C R464H is a pathogenic mutation, here we provide additional functional evidence that the CCDC88C R464H activated the JNK-caspase pathway in the primary neuronal cells derived from the mammalian brain. We used the day-18 rat embryonic cortical neurons as our experimental cell model. Similar to the results we previously obtained from the human HEK293 cells, overexpression of the CCDC88C R464H induced the JNK hyperphosphorylation and caspase-3 cleavage in rat primary cortical neurons (Figure 2). This result therefore strengthened our hypothesis that CCDC88C R464H triggers apoptosis and contributes to the cerebellar atrophy in SCA40 patients.

[1] https://macarthurlab.org/2014/11/18/a-guide-to-the-exome-aggregation-consortium-exac-data-set/

Table 1--Screening of autosomal dominant Spinocerebellar ataxia and Spastic paraplegia markers in population genetics databases.

Figure 1--CCDC88C variants around Arg-464 position in 1000 Genome Phase III.

Figure 2--Overexpression of the CCDC88C R464H DNA construct induced JNK hyperphosphorylation and caspase-3 cleavage in rat primary cortical neuronal cells. Two independent trials (Set1 and Set2) were performed and the results are consistent.

References

1. Tsoi,H., Yu,A.C.S., Chen,Z.S., Ng,N.K.N., Chan,A.Y.Y., Yuen,L.Y.P., Abrigo,J.M., Tsang,S.Y., Tsui,S.K.W., Tong,T.M.F., et al. (2014) A novel missense mutation in CCDC88C activates the JNK pathway and causes a dominant form of spinocerebellar ataxia. J. Med. Genet., 51, 590-5.

2. Cadieux-Dion,M., Turcotte-Gauthier,M., Noreau,A., Martin,C., Meloche,C., Gravel,M., Drouin,C.A., Rouleau,G.A., Nguyen,D.K. and Cossette,P. (2014) Expanding the clinical phenotype associated with ELOVL4 mutation: study of a large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia. JAMA Neurol., 71, 470-5.

3. Lek,M., Karczewski,K.J., Minikel,E. V., Samocha,K.E., Banks,E., Fennell,T., O'Donnell-Luria,A.H., Ware,J.S., Hill,A.J., Cummings,B.B., et al. (2016) Analysis of protein-coding genetic variation in 60,706 humans. Nature, 536, 285-291.

4. Pollak,A., Skorka,A., Mueller-Malesi?ska,M., Kostrzewa,G., Kisiel,B., Waligora,J., Krajewski,P., O?dak,M., Korniszewski,L., Skarzy?ski,H., et al. (2007) M34T and V37I mutations in GJB2 associated hearing impairment: evidence for pathogenicity and reduced penetrance. Am. J. Med. Genet. A, 143A, 2534-43.

5. Mannan,A.U. (2008) Response to Martignoni et al. Am. J. Hum. Genet., 83, 128-130.

6. Smeets,C.J.L.M. and Verbeek,D.S. (2016) Reply: SCA23 and prodynorphin: is it time for gene retraction? Brain, 139, e43.

7. Ham,J., Babij,C., Whitfield,J., Pfarr,C.M., Lallemand,D., Yaniv,M. and Rubin,L.L. (1995) A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death. Neuron, 14, 927-39.

8. Yang,D.D., Kuan,C.Y., Whitmarsh,A.J., Rincon,M., Zheng,T.S., Davis,R.J., Rakic,P. and Flavell,R.A. (1997) Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature, 389, 865-70.

9. Kuan,C.Y., Yang,D.D., Samanta Roy,D.R., Davis,R.J., Rakic,P. and Flavell,R.A. (1999) The Jnk1 and Jnk2 protein kinases are required for regional specific apoptosis during early brain development. Neuron, 22, 667-76.

10. Nagasaki,M., Yasuda,J., Katsuoka,F., Nariai,N., Kojima,K., Kawai,Y., Yamaguchi-Kabata,Y., Yokozawa,J., Danjoh,I., Saito,S., et al. (2015) Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals. Nat. Commun., 6, 8018.

11. Francioli,L.C., Menelaou,A., Pulit,S.L., van Dijk,F., Palamara,P.F., Elbers,C.C., Neerincx,P.B.T., Ye,K., Guryev,V., Kloosterman,W.P., et al. (2014) Whole-genome sequence variation, population structure and demographic history of the Dutch population. Nat. Genet., 46, 818-825.

12. Teo,Y.-Y., Sim,X., Ong,R.T.H., Tan,A.K.S., Chen,J., Tantoso,E., Small,K.S., Ku,C.-S., Lee,E.J.D., Seielstad,M., et al. (2009) Singapore Genome Variation Project: a haplotype map of three Southeast Asian populations. Genome Res., 19, 2154-62.

13. Walter,K., Min,J.L., Huang,J., Crooks,L., Memari,Y., McCarthy,S., Perry,J.R.B., Xu,C., Futema,M., Lawson,D., et al. (2015) The UK10K project identifies rare variants in health and disease. Nature, 526, 82-90.

14. Yasser,S., Fecto,F., Siddique,T., Sheikh,K.A. and Athar,P. (2010) An unusual case of familial ALS and cerebellar ataxia. Amyotroph. Lateral Scler., 11, 568-70.

15. Li,H. and Durbin,R. (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25, 1754-60.

16. McKenna,A., Hanna,M., Banks,E., Sivachenko,A., Cibulskis,K., Kernytsky,A., Garimella,K., Altshuler,D., Gabriel,S., Daly,M., et al. (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res., 20, 1297-303.

17. Li,M.-X., Gui,H.-S., Kwan,J.S.H., Bao,S.-Y. and Sham,P.C. (2012) A comprehensive framework for prioritizing variants in exome sequencing studies of Mendelian diseases. Nucleic Acids Res., 40, e53.

Conflict of Interest:

None declared

Response to e letter ID jmedgenet el; 2826 by Refaeli et al., dated June14, 2016 A considerable amount of literature on the role of podocalyxin-like (PODXL) protein in normal mammalian kidney functions and to a lesser extent in mouse brain development is available. We agree that these studies particularly, "Anuria, Omphalocele, and Perinatal Lethality in Mice Lacking the Cd34-Related Protein Podocalyxin" by Doyonnas et al (1) and "Podocalyxin is a Novel Polysialylated Neural Adhesion Protein with Multiple Roles in Neural Development and Synapse Formation" by Vitureira et al (2) using podxl -/- mouse embryos and newborns have unequivocally demonstrated the role of this gene in renal function and brain development respectively. Doyonnas et al., (1) in their paper do mention that i) anuria and renal failure is evidenced only postnatally as there is possible maternal clearance in the embryos; ii) there is no evidence of extensive proteinuria, which is characteristic of leaky podocyte filtration in human nephrotic syndromes; and iii) more importantly, that there were no developmental anomalies in the hematopoietic and vascular endothelial cells which also express high levels of podocalyxin. The last observation, authors hypothesized, may have been enabled through functional compensation by other sialomucins such as CD34. Data on the status of neural development in their newborn mice with identical genetic background, which would have proved the effect of podxl deficiency in these two vital processes is however missing in their paper. Overall, the results from these mouse studies suggest that i) podxl protein is almost ubiquitous in expression, multifunctional, may have varying interacting partners and that functional/genetic redundancy exists. In our opinion, these features of PODXL are exactly what the two mutation studies in this gene one on autosomal dominant familial focal and segmental glomerulosclerosis (FSGS) (3) and our study on autosomal recessive juvenile parkinsonism (4) reflect. This is not unexpected considering the different genetic backgrounds in different families. Notably, in the paper by Barua et al, (3) i) the incomplete penetrance of the private variant p.L442R (i.e., one individual carrying the mutation but asymptomatic even at 53 years of age); ii) varying age of onset ranging from early teenage to adulthood but developing end stage renal disease between 2nd to 6th decade of life; iii) varying disease severity across affected individuals in the family but certainly not post- natal/newborn mortality (unlike in mice); and iv) rarity of mutations in PODXL gene in 176 additional FSGS families; together with mutant PODXL protein characterization in vitro which showed that the index mutation results in dimerization but does not alter protein stability, extracellular domain glycosylation, cell surface expression, global subcellular localisation and interactions with its intracellular binding partner ezrin are all the features of PODXL gene that need to be kept in mind prior to assigning it an indispensable role in kidney function and survival in humans. It is indeed appropriate that Barua et al (3) conclude that rare variants with low statistical genetic evidence such as observed in their study do not contribute significantly to glomerular disease in humans.

Based on these observations, it is not unlikely that the index family with ARJP with homozygous mutations predicted to result in loss of protein did not manifest any symptoms of FSGS or any other kidney dysfunction, based on the data of last clinical examination in 2009. As for the two other concerns of Refaeli et al on the index mutation being unlikely of germline origin and NGS errors, both can be disregarded as both the parents are heterozygous for the index mutation (data not shown), and all informative NGS data are validated by Sanger sequencing. Further, their concern that mutations identified in unrelated PD cohort are just common protein altering variants is also ruled out as these were not observed in either our population or reported in any publically available large databases and therefore, were functionally characterized in our study. As for the demonstration of absence of PODXL RNA or protein, we have not been able to do that due to non-availability of tissues. It may please be noted that in the absence of clinical indications, it is difficult to obtain ethical committee clearance for collection of target tissues. In summary, from careful interpretation of the findings in the above mentioned studies as well as abundant data in literature [for eg. See Liao and Zhang, PNAS, 2008 (5)], it is clear that there are notable differences between mice and humans in i) the developmental phenomena; ii) functional compensatory mechanisms/genetic redundancy; iii) gene-gene interactions; iv) tissue specific transcript expression etc. Though experimental evidence for most of these aspects with specific reference to PODXL is currently unavailable, such differences may be believed to be useful to explain pleiotropy in PODXL [for eg. similar to that documented in cystic fibrosis (6)], possible tolerance to null alleles enabling apparently normal kidney function as witnessed in the ARJP family in our study (4) and comparatively late age of onset of FSGS, incomplete penetrance of the gene mutation etc. (3) and several other associated features. Finally, we would like to thank Rafaeli et al for their interest in our work and giving us an opportunity to highlight some of the differences which may exist between humans and mice in general and for role of podocalyxin in particular. We firmly believe that findings from animal models of human diseases are powerful tools but are not always true representatives for understanding complete human health and disease biology. Additional data as and when available on PODXL associated human phenotypes would be most insightful for our enhanced understanding of the role of PODXL in humans. References: 1. Doyonnas R, Kershaw DB, Duhme C, et al. Anuria, omphalocele, and perinatal lethality in mice lacking the CD34-related protein podocalyxin. J Exp Med 2001;194(1):13-27. 2. Vitureira N, Andres R, Perez-Martinez E, et al. Podocalyxin is a novel polysialylated neural adhesion protein with multiple roles in neural development and synapse formation. PLoS One 2010;5(8):e12003.? 3. Barua M, Shieh E, Schlondorff J, et al. Exome sequencing and in vitro studies identified podocalyxin as a candidate gene for focal and segmental glomerulosclerosis. Kidney Int 2014;85(1):124-33. 4. Sudhaman S, Prasad K, Behari M, et al. Discovery of a frameshift mutation in podocalyxin-like (PODXL) gene, coding for a neural adhesion molecule, as causal for autosomal-recessive juvenile Parkinsonism. J Med Genet 2016; 53(7):450-6. 5. Liao B, Zhang J. Null mutations in human and mouse orthologs frequently result in different phenotypes. PNAS 2008;105(19):6987-92. 6. Sing C, Risser D, Howatt W, Erickson R. Phenotypic heterogeneity in cystic fibrosis. American Journal of Medical Genetics 1982;13:179-195.

Conflict of Interest: None declared

Dr. Sumedha Sudhaman1 Prof. Kameshwar Prasad2 Prof. Madhuri Behari2 Dr. Uday B Muthane3 Dr. Ramesh C Juyal4 Prof. BK Thelma1 1Department of Genetics, University of Delhi South Campus, New Delhi, India 2Department of Neurology, All India Institute of Medical Sciences, New Delhi, India 3Parkinson's and Ageing Research Foundation, Bengaluru, Karnataka, India 4Regional Center for Biotechnology, Faridabad, Haryana, India

28 July, 2016

Conflict of Interest:

None declared

I just read the articles on SMA1, SMA2, SMA3. I felt it important to tell you of my son, born 1/26/70. When I took him to his first checkup at 3 months old, I voiced my concern for his floppy head. Again at 6 months old, when he couldn't sit up, roll over, kick against resistance. Finally, the Pediatrician agreed to get an appointment with Yale Hospital in CT at 8 mos. The biopsy was shown to be SMA 1. We weren't given any chance of survival. He was subsequently re-diagnosed at 3 years old, and again the results were, SMA1. He is now 46 years old. Graduated UCONN with honors and is still living in Litchfield CT. He is not on a ventilator but uses a bi-pap, 6 to 8% lung capacity. We have been told over and over there is no medication and there is no trials he could be involved with. However, with the social media being his way to communicate with the mass population he has discovered the Salbutamol available now through a client with SMA2. She has given us hope that this new drug will give him even the slightest increase in strength to keep the two fingers he uses to control his fiber optic wheelchair. Unfortunately its too expensive. It is heartbreaking to come so close to possibly gaining enough strength to stay mobile. If there is any possible way he could be helpful to your programs via computer please let us know. Respectfully, June Lajoie Strada

Conflict of Interest:

None declared

Dear Editor

Many articles written on the age of survival of babies with trisome 13 and 18 fail to factor in the influence of modern intensive care treatment modalities.

Use of artificial respirators, suctioning of respiratory secretons coupled with supplemental oxygen and nasogastric tube feedings may prolong survival.For the survivors beyond 7-10 days, it has become routine to feed these babies with tube feedings in US and even a gastrostomy is done. Gastroesophageal reflux is treated with pharmacotherapy.

In other words, are these survival tables truly reflective of the natural history of these trisomies? We need these factors included in the survival tables in order to use them for genetic counselling. Perhaps survival data from countries which do not provide such modern treatments would be useful.