Letter to Editor Shaheen et al. 1 recently reported a homozygous 5 bps deletion mutation (c.1299_1303delTAAG; p.Phe433Leufs*6) in the human polo-like kinase 4 (PLK4,MIM 605031) gene and proposed that it is a compelling candidate for primordial dwarfism (PD). Autozygosity mapping and LOD score, method of estimating linkage distances, are adopted for the novel locus identification. Candidate gene hunting among 144 in the region is based on filtering through ToppGene suite 2. Mutation screening through Sanger sequencing of PLK4 gene is only based on ToppGene suite ranking. In this study authors have not mentioned how many and which samples were selected for genotyping and linkage analysis so the predicted maximum LOD score cannot be calculated. Maximum multipoint LOD score (2.5) obtained by the authors is quite lower than the established threshold values (3.0). In article it has been stated that all PD genes in training set in comparison to all 144 genes of the locus in test set were enrolled in ToppGene candidate prioritization. These genes lists must be provided along with test parameters. In our opinion these informations are essential for data reproducibility and validation. Only PLK4 gene has been selected for Sanger sequencing but The candidate region (Chr4:112904466-129392060, GRCh37/hg19) includes some of the well- known syndromes characterized by autosomal recessive intellectual disabilities, microcephaly, short stature, and overlapping phenotypes. Alazami syndrome caused by mutations in LARP7, autosomal recessive type 1 mental retardation caused by mutations in PRSS12, Bardet-Biedl syndrome caused by mutations in BBS7 and BBS12, Van Maldergem syndrome 2 caused by mutations in FAT4, neuronal ceroid lipofuscinosis 7 caused by mutations in MFSD8, a multisystem disorder including brain function caused by mutation in SCLT1, as well as genes involved in mitosis and brain development e.g. MAD2L1, FGF2, INTU, have not been discussed throughout the manuscript. Furthermore, chromatograms for obligate carriers have not been presented nor the Saudi population screening for minor allele frequency has been performed. Similarly a homozygous variant (NM_014264.4, c.1556G>C; p.Trp519Ser) reported in ESP6500 (http://evs.gs.washington.edu/EVS/) alters a highly conserved amino acid of PLK4 however it has not been assigned to any of primordial dwarfism phenotype and not discussed by the authors. In fact, phenotypic overlaps among the study mutants and the above syndromes would be of far more value instead of paying an exaggerated attention to PLK4 biology. References

1 Shaheen R, Al Tala S, Almoisheer A et al. Mutation in PLK4, encoding a master regulator of centriole formation, defines a novel locus for primordial dwarfism. Journal of medical genetics 2014. 2 Chen J, Bardes EE, Aronow BJ et al. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic acids research 2009; 37: W305-11.

Conflict of Interest:

None declared

C-terminus truncation of Seipin as a good potential sign of diseases

Wei-Li Yu and Yun Sun

Seipin is a transmembrane protein of the endoplasmic reticulum with N, C-terminus residing the intracytoplasmic side.1 2 Loss of Seipin function has been shown to lead to severe lipodystrophy, motor neuropathy and silver syndrome.3 However, its molecular mechanism is still unknown. Recently Guillén-Navarro E et al 4 indicated that complete skipping of exon 7 was associated with a new neurodegenerative syndrome. The mutation caused early termination, resulting in C-terminally truncated proteins p.Tyr289LeufsX64. It demonstrated that C-terminus of Seipin played a key role in the pathogenesis of this new neurological disease. This result suggests that C-terminus truncation of Seipin might be a good potential prediction of this disease. Other studies have been carried with the similar aim. Van Maldergem et al 5 had reported that C-terminus deficiency of Seipin due to kipping of exon 7 caused a case of severe psychomotor delay and pyramidal signs. These mutations were associated with intellectual impairment. Furthermore, Yang W et al 6 found that C-terminus truncated proteins such as R275X and Q391X, led to lipodystrophy. It indicated that C-terminus of Seipin could regulate lipogenesis and adipogenesis. In addition, Yang W et al 7 identified that C-terminus of Seipin interacted with 14-3-3β, which patiotemporally recruited an interacting partner of actin-severing protein cofilin-1 during adipocyte differentiation. They proposed that C-terminus of Seipin might regulate adipogenesis by recruiting cofilin-1 to remodel actin cytoskeleton through the 14-3-3β protein.

These findings suggest that C-terminus truncation of Seipin may be a potential sign of diseases such as lipodystrophy, psychomotor delay and neurological diseases. We greatly enjoyed reading the article by Guillén-Navarro E et al and believe C-terminus truncation of Seipin may be useful for prediction of diseases.

The authors declare no conflict of interest.

1. Magré J, Delépine M, Khallouf E, Gedde-Dahl T Jr, Van Maldergem L, Sobel E, Papp J, Meier M, Mégarbané A, Bachy A, Verloes A, d’Abronzo FH, Seemanova E, Assan R, Baudic N, Bourut C, Czernichow P, Huet F, Grigorescu F, de Kerdanet M, Lacombe D, Labrune P, Lanza M, Loret H, Matsuda F, Navarro J, Nivelon-Chevalier A, Polak M, Robert JJ, Tric P, Tubiana-Rufi N, Vigouroux C, Weissenbach J, Savasta S, Maassen JA, Trygstad O, Bogalho P, Freitas P, Medina JL, Bonnicci F, Joffe BI, Loyson G, Panz VR, Raal FJ, O’Rahilly S, Stephenson T, Kahn CR, Lathrop M, Capeau J; BSCL Working Group. Identification of the gene altered in Berardinelli-Seip congenital lipodystrophy on chromosome 11q13. Nat Genet 2001; 28: 365-70. 2. Lundin C, Nordstrom R, Wagner K, Windpassinger C, Andersson H, von Heijne G and Nilsson I. Membrane topology of the human seipin protein. FEBS Lett 2006; 580: 2281-2284. 3. Chen W, Yechoor VK, Chang BH, Li MV, March KL, Chan L. The human lipodystrophy gene product Berardinelli-Seip congenital lipodystrophy 2/seipin plays a key role in adipocyte differentiation. Endocrinology 2009; 150:4552-4561. 4. Encarna Guillén-Navarro, Sofía Sánchez-Iglesias, Rosario Domingo-Jiménez, Berta Victoria, Alejandro Ruiz-Riquelme, Alberto Rábano, Lourdes Loidi, Andrés Beiras, Blanca González-Méndez, Adriana Ramos, Vanesa López-González, María Juliana Ballesta-Martínez, Miguel Garrido-Pumar, Pablo Aguiar, Alvaro Ruibal, Jesús R Requena, and David Araújo-Vilar. A new seipin-associated neurodegenerative syndrome. J Med Genet 2013; 50:401-409. 5. Van Maldergem L, Magré J, Khallouf TE, Gedde-Dahl T Jr, Delépine M, Trygstad O, Seemanova E, Stephenson T, Albott CS, Bonnici F, Panz VR, Medina JL, Bogalho P, Huet F, Savasta S, Verloes A, Robert JJ, Loret H, De Kerdanet M, Tubiana-Rufi N, Mégarbané A, Maassen J, Polak M, Lacombe D, Kahn CR, Silveira EL, D’Abronzo FH, Grigorescu F, Lathrop M, Capeau J, O’Rahilly S. Genotype-phenotype relationships in Berardinelli-Seip congenital lipodystrophy. J Med Genet 2002; 39: 722-33. 6. Yang W, Thein S, Guo X, Xu F, Venkatesh B, Sugii S, Radda GK and Han W. Seipin differentially regulates lipogenesis and adipogenesis through a conserved core sequence and an evolutionarily acquired C-terminus. Biochem J 2013; 452: 37-44. 7. Yang W, Thein S, Wang X, Bi X, Ericksen RE, Xu F, Han W. BSCL2/seipin regulates adipogenesis through actin cytoskeleton remodeling. Human Molecular Genetics 2014; 23: 502-513.

Conflict of Interest:

None declared

RSS was first described in 1953 [1]. It is a clinically and genetically heterogeneous condition. Clinical diagnosis is based on various characteristics including slow growth before and after birth, relative macrocephaly, a prominent forehead at a young age, hemihypotrophy, and fifth finger clinodactyly. Five different diagnostic scoring systems of RSS have been published. These systems take into account most major clinical features and sometimes minor clinical features, and are on the whole quite similar. These five scoring systems were recently compared in the Journal of Medical Genetics in terms of sensitivity, specificity, and positive or negative predictive value [2]. In this evaluation, the scoring system described by Netchine et al. [3] showed a high degree of specificity and sensitivity. This scoring system has also been used by other teams from various countries in the diagnosis of RSS (see for instance [4]). Furthermore, the article describing this scoring system [3] was reviewed by the editorial board of The Journal of Clinical Endocrinology & Metabolism (JCEM) and received an award in 2008 from the Endocrine Society. This article has been cited in PUBMED 128 times. The first described molecular abnormality in RSS was the maternal uniparental disomy of chromosome 7 (mUPD7). This defect is present in 5- 10% of individuals affected by RSS [5, 6]. This observation suggests the aberrant dosage of imprinted genes may be involved in the pathology of RSS, although the gene(s) involved remain unknown. Recently, abnormalities of 11p15, a locus also harboring imprinted genes, have been described. This region contains two separate imprinting clusters: the telomeric domain (ICR1) that regulates H19 and IGF2, and the centromeric domain (ICR2) that regulates the long non-coding RNA KCNQ1OT1 and CDKN1C genes. Of note, cytogenetic, genetic, and epigenetic disturbances of the 11p15 region encompassing ICR1 and ICR2 are associated with the overgrowth disorder Beckwith Wiedemann syndrome (BWS). BWS is a clinical and molecular mirror of RSS, and around 8% of cases involve loss of function mutations of CDKN1C [7]. In RSS, loss of methylation (LOM) of the paternal ICR1 is the most prevalent defect. This epigenetic change is found in up to 60% of RSS patients [3, 8], although it has been reported to occur less frequently in some populations (e.g. with a frequency of ~31% in Japanese patients) [4]. Approximately 30% of RSS patients are ? idiopathic ? with no molecular anomaly yet identified.

General comments on the items of the scoring system described in [3]: Young children with RSS have a very different clinical profile than older children or adults with RSS. Feeding difficulties are much more pronounced in early life and therefore have to be assessed at a young age. Older children and adults may become overweight. Feeding difficulties can be difficult to assess; therefore, one of the conclusions of our paper in 2007 was to replace the item ? feeding difficulties ? with ? feeding difficulties and /or a BMI two SDs below the mean at around two years of age ?. The prominent forehead is distinct from relative macrocephaly. Indeed, relative macrocephaly is characterized by a growth restriction affecting mostly the body, whereas circumference of the head is relatively less unaffected. A prominent forehead however, is a dysmorphic feature that describes the characteristic shape of these patients' forehead protruding over the plane of the face. This feature tends to disappear in late childhood and therefore has to be assessed around two years of age. Body asymmetry is a feature of RSS, but is not present in all RSS patients. This feature is more frequent in RSS patients with epigenetic changes to 11p15 than in patients with mUPD7. For example, in our sample of 145 RSS patients with 11p15 LOM, 79% of patients had hemihypotrophy, whereas in a sample of 16 RSS patients with mUPD7, none of the patients were affected by hemihypotrophy (p<0.0001). This is probably due to the fact that the 11p15 LOM occurs in a mosaic manner, such that the proportion of cells that possess this epigenetic change may differ between the left and right side of the body. The item post natal growth retardation also has to be assessed around two years of age. This is because most RSS patients are treated with growth hormone generally starting between two and four years of age, to improve their growth rate. Bone age and pubertal status also has to be taken into account when analyzing the growth of a child affected by RSS. Indeed, these children frequently have an advanced bone age due to aggressive adrenarche and early puberty. Thus, even though their height may be normal during childhood, their final height may be lower than average due to premature bone fusion.

Specific comments in response to this letter The diagnosis of RSS in the patients reported by Brioude et al. is not questionable because all the patients carrying the mutation fulfilled the clinical criteria of RSS as described by Netchine et al. in 2007. Furthermore, these patients also fulfill the RSS clinical criteria according to other validated RSS clinical scoring systems such as the Birmingham scoring system [2]. However, as mentioned above, the clinical diagnosis of RSS is often difficult. RSS scoring systems are helpful to orient the diagnosis; nonetheless, it is also very important that an experienced clinical geneticist or pediatrician make this diagnosis. The pediatrician (IO) who referred this family to our laboratory has a lot of clinical experience in RSS and has followed two generations of this family (second and third). The pediatrician (IO) became convinced of the diagnosis of a very rare case of familial RSS when the third generation of this family came to the clinic for consultation. In the clinical scoring system described by Netchine et al. [3], body asymmetry is one of the six criteria for the clinical diagnosis of RSS. However, in a family carrying a CDKN1C mutation, body asymmetry is not expected to occur because the molecular defect is present in all the cells of the body (unlike the mosaic epigenetic change at the 11p15 locus). This hypothesis is in accordance with the observation that patients with Beckwith Wiedemann syndrome who carry CDKN1C loss of function mutations are not usually affected by body asymmetry [7]. Similarly, patients with IMAGe syndrome with a mutation in CDKN1C do not have body asymmetry [9]. Thus, the absence of body asymmetry does not exclude the diagnosis of RSS in this particular case. Furthermore, we found that RSS patients with mUPD7 do not show body asymmetry, and some RSS patients with 11p15 LOM are also unaffected (20% in our sample of patients). As mentioned above, all the patients described here had feeding difficulties and/or a BMI two SDs below the mean at around two years of age. We can consider a low BMI to be equivalent to feeding difficulties, as mentioned in the legend of table 2 and proposed in our paper in 2007 [3]. Relative macrocephaly was not present at birth for all the patients presented here (notably in the propositus IV.1). However, this trait was present at two years of age in all patients carrying the mutation. Therefore, this characteristic may help to distinguish RSS patients with a mutation in CDKN1C from patients with 11p15 LOM. However, the absence of relative macrocephaly is not enough to preclude the clinical diagnosis of RSS in these patients. Although the triangular face does not appear to be obvious in the pictures of the patients, this feature has been confirmed by the patients' physician (IO). Furthermore, this trait (triangular face and prominent forehead) is usually less obvious in adult patients. Besides, a triangular face is not a clinical criterion for RSS diagnosis and is not part of our scoring system. All the patients described here presented growth retardation: Length at birth and then height and/or weight was two SDs below the mean according to French reference charts both at birth and after birth. All the patients with the CDKN1C mutation did not attain a normal height at the age of 2 years. Patient III.7 received a long-term therapy consisting of growth hormones and Triptorelin acetate and thus reached a final height of 157 cm. However this patient's sister, (patient III.6) received growth hormone therapy for a shorter duration than her sister resulting in a lower final height. Today, most RSS patients are treated with growth hormones and the best improvement in final height is attained when the rapid advancement in bone age is also treated. Individuals II.4 and III.10 do not carry the CDKN1C mutation, and cannot be considered as having RSS because they do not present the clinical criteria as described above. These two individuals did not show relative macrocephaly at birth or at the age of two years. Their size at birth was either normal (II.4), or, in the case of III.10, small, but nonetheless larger than new-born babies corresponding to family members who carry the mutations. Individuals II.4 and III.10 had a normal BMI, and II.4 had a normal adult height without any therapy. We agree with the point that individual III.10 showed a short final height despite growth hormone therapy. This patient was followed like the other patients with short stature of this family by the same pediatrician (IO), who did not clinically assess her as having RSS. To date, the causal mechanism of her short adult height has not been elucidated.

CONCLUSION Defining the clinical criteria of a clinically and molecularly heterogeneous syndrome like RSS is not straightforward. Nonetheless, we are convinced that the family described here, represents a rare case of familial RSS. The diagnosis of RSS was made by a physician experienced in RSS who had the opportunity to follow and treat two generations of this exceptional family. The scoring system described by Netchine et al. [3], but also other RSS scoring systems such as those evaluated by Dias et al. [2] also favor this diagnosis. The absence of body asymmetry does not rule out the diagnosis of RSS, as in mUPD7 RSS cases or in 20% of 11p15 LOM cases. In this case, the absence of body asymmetry is consistent with the genetic cause identified. This CDKN1C mutation is therefore a new genetic cause of familial RSS. The CDKN1C mutation identified in this RSS familial case affects the same amino acid as a mutation described by Arboleda et al. [9] in patients with IMAGe syndrome. This observation will be of great interest for the readers of The Journal of Medical Genetics. IMAGe syndrome has some similarities with RSS, including fetal and postnatal growth retardation and a prominent forehead, but is also typically characterized by bone anomalies and a severe adrenal insufficiency that were not present in this family. It is striking to note that a mutation on the exact same amino acid, but with a different substitution, can lead to severe adrenal insufficiency in one case and a normal adrenal function in the other case, and therefore to two distinct syndromes. As of 2013, 60 years after the first description of RSS, several molecular causes of RSS have been identified (mUPD7 and various types of 11p15 anomalies) accounting for about 70% of etiologies. We suggest that CDKN1C mutations are a new molecular cause of familial RSS. Given the clinical and molecular heterogeneity of RSS we suggest that RSS should no longer be considered as a syndrome but as a spectrum of disorders with a variety of causes. This would make it easier to understand why RSS is clinically and molecularly heterogeneous. However, it is important for RSS patients to be identified as belonging to a same spectrum and not to multiple categories of very rare conditions, because these patients share a lot of clinical manifestations and have similar treatment needs. This will allow the development of common clinical guidelines for these patients, and well help in the implementation of clinical trials that are always difficult to establish for rare diseases. The identification of exact genetic defect is also a cornerstone for an accurate genetic counseling.

1 Silver HK, Kiyasu W, George J, Deamer WC. Syndrome of congenital hemihypertrophy, shortness of stature, and elevated urinary gonadotropins. Pediatrics 1953;12(4):368-76. 2 Dias RP, Nightingale P, Hardy C, Kirby G, Tee L, Price S, Macdonald F, Barrett TG, Maher ER. Comparison of the clinical scoring systems in Silver -Russell syndrome and development of modified diagnostic criteria to guide molecular genetic testing. J Med Genet 2013;50(9):635-9. 3 Netchine I, Rossignol S, Dufourg MN, Azzi S, Rousseau A, Perin L, Houang M, Steunou V, Esteva B, Thibaud N, Demay MC, Danton F, Petriczko E, Bertrand AM, Heinrichs C, Carel JC, Loeuille GA, Pinto G, Jacquemont ML, Gicquel C, Cabrol S, Le Bouc Y. 11p15 imprinting center region 1 loss of methylation is a common and specific cause of typical Russell-Silver syndrome: clinical scoring system and epigenetic-phenotypic correlations. J Clin Endocrinol Metab 2007;92(8):3148-54. 4 Fuke T, Mizuno S, Nagai T, Hasegawa T, Horikawa R, Miyoshi Y, Muroya K, Kondoh T, Numakura C, Sato S, Nakabayashi K, Tayama C, Hata K, Sano S, Matsubara K, Kagami M, Yamazawa K, Ogata T. Molecular and clinical studies in 138 Japanese patients with Silver-Russell syndrome. PLoS One 2013;8(3):e60105. 5 Kotzot D, Schmitt S, Bernasconi F, Robinson WP, Lurie IW, Ilyina H, Mehes K, Hamel BC, Otten BJ, Hergersberg M, et al. Uniparental disomy 7 in Silver-Russell syndrome and primordial growth retardation. Hum Mol Genet 1995;4(4):583-7. 6 Preece MA, Price SM, Davies V, Clough L, Stanier P, Trembath RC, Moore GE. Maternal uniparental disomy 7 in Silver-Russell syndrome. J Med Genet 1997;34(1):6-9. 7 Brioude F, Lacoste A, Netchine I, Vazquez MP, Auber F, Audry G, Gauthier -Villars M, Brugieres L, Gicquel C, Le Bouc Y, Rossignol S. Beckwith- Wiedemann Syndrome: Growth Pattern and Tumor Risk according to Molecular Mechanism, and Guidelines for Tumor Surveillance. Horm Res Paediatr 2013;80:457-65. 8 Gicquel C, Rossignol S, Cabrol S, Houang M, Steunou V, Barbu V, Danton F, Thibaud N, Le Merrer M, Burglen L, Bertrand AM, Netchine I, Le Bouc Y. Epimutation of the telomeric imprinting center region on chromosome 11p15 in Silver-Russell syndrome. Nat Genet 2005;37(9):1003-7. 9 Arboleda VA, Lee H, Parnaik R, Fleming A, Banerjee A, Ferraz-de-Souza B, Delot EC, Rodriguez-Fernandez IA, Braslavsky D, Bergada I, Dell'angelica EC, Nelson SF, Martinez-Agosto JA, Achermann JC, Vilain E. Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome. Nat Genet 2012;44(7):788-92.

Conflict of Interest:

None declared

Re: Disruption of RAB40AL function leads to Martin-Probst syndrome, a rare X-linked multisystem neurodevelopmental human disorder. Bedoyan JK, Schaibley VM, Peng W, Bai Y, Mondal K, Shetty AC, Durham M, Micucci JA, Dhiraaj A, Skidmore JM, Kaplan JB, Skinner C, Schwartz CE, Antonellis A, Zwick ME, Cavalcoli JD, Li JZ, Martin DM. J Med Genet. 2012 May;49(5):332- 40.

With great interest we have read the article by Bedoyan and colleagues. The authors state that disruption of RAB40AL function by a dinucleotide missense change (chrX:102,079,078-102,079,079AC?GA p.D59G; hg18) causes Martin-Probst syndrome, a rare X-linked disorder characterised by deafness, cognitive impairment, short stature and distinct craniofacial dysmorphisms, among other clinical features. However, we have reasons to question that conclusion.

We have identified the same dinucleotide missense change in four out of 446 index patients from families with X-linked intellectual disability (XLID) by performing high-throughput sequencing of all X chromosome- specific exons. However, one patient in whom we had identified the p.D59G substitution also carried a deletion on chromosome 14 that unequivocally explained the clinical phenotype, which led us to speculate that in this family the RAB40AL change may not be causative of the phenotype. Therefore, we carried out segregation analysis in the remaining three families. One family was not informative for the RAB40L change. In this family both the clinical phenotype and segregation analyses of additionally identified variants suggested that this family more likely carries a pathogenic mutation in another XLID gene. In the remaining two families, Sanger sequencing confirmed the RAB40AL p.D59G substitution in the index patients. However, in each of these families, one of the affected male sibs did not carry the p.D59G substitution. Conversely, the exome variant server (ESP6500) and dbSNP database report two normal males who are hemizygous for the p.D59G substitution (dbSNP134 rs145606134). In yet another XLID family, we have observed a frameshift mutation (chrX:102192671-102192672 [hg19], ins1bp, p.A143GfsX12) in RAB40AL. In this family, in a healthy hemizygous male as well as several healthy homozygous females carried the frameshift variant.

Taken together, these data seem to indicate that the RAB40AL variants observed are not related to the XLID in our families. Moreover, they suggest that the RAB40AL p.D59G substitution may not be the cause of Martin-Probst syndrome but may represent a rare benign variant. While it is not trivial to prove that a given variant is NOT involved in XLID, the aggregate evidence presented here suggests that the role of RAB40AL in XLID and in particular, that of the p.D59G substitution in Martin-Probst syndrome should be revisited.

Conflict of Interest:

None declared