Dr Gajja S Salomons
email: g.salomons@vumc.nl

Efraim H Rosenberg

Ligia S Almeida

Tjitske Kleefstra

Charles E Schwartz

Vassili Valayannopoulos

Omar A Abdul-Rahman

Nicola Poplawski

Laura Vilarinho

Philipp Wolf

Ofir T Betsalel

Johan Den Dunnen

Cornelis Jakobs

) Charles

E Schwartz

Characterization of novel SLC6A8 variants with the use of splice-site analysis tools and implementation of a newly developed LOVD database

Keywords: SLC6A8, XLMR, Splicing, LOVD

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In 2001, the X-linked mental retardation (XLMR) syndrome, SLC6A8 deficiency, was identified due to a creatine deficiency in the brain caused by mutations in the creatine transporter (SLC6A8) gene (MIM 300036). SLC6A8 has been mapped to Xq28 [START_REF] Gregor | Assignment of the creatine transporter gene (SLC6A8) to human chromosome Xq28 telomeric to G6PD[END_REF] and is a member of the solute-carrier family 6 (neurotransmitter transporters). The clinical presentation of males affected with SLC6A8 deficiency is mental retardation (MR), expressive speech and language delay, epilepsy, developmental delay and autistic behavior. Laboratory hallmarks include a reduction of the creatine signal in the proton magnetic resonance spectroscopy (H-MRS) of the brain, an increased urinary creatine/creatinine ratio and impaired creatine uptake in cultured fibroblasts. In female carriers, learning disabilities of varying degrees have been noted. Two studies of males with X-linked MR estimated the prevalence of SLC6A8 deficiency to be 2.1% (CI: 0.44 -3.76) 2 and 1.5% (CI: 0 -4.46). [START_REF] Betsalel | Detection of low-level somatic and germline mosaicism by denaturing high-performance liquid chromatography in a EURO-MRX family with SLC6A8 deficiency[END_REF] A third study of males with MR revealed a prevalence of 0.8% (CI: 0.02 -1.7%). [START_REF] Clark | X-linked creatine transporter (SLC6A8) mutations in about 1% of males with mental retardation of unknown etiology[END_REF] Within the last decade many novel variants in the SLC6A8 gene have been detected. These variants are difficult to classify as pathogenic or non disease causing since the variants are either located in the coding region, but are synonymous, or are found in the intronic regions. However, such variants may affect proper splicing and are potentially pathogenic. [START_REF] Salomons | An intriguing "silent" mutation and a founder effect in antiquitin (ALDH7A1)[END_REF] We therefore not only tested if such variants could be properly analyzed by studying previously classified variants, but also subjected 28 novel variants to these tools. Moreover, to facilitate worldwide diagnostic and research laboratories interested in the SLC6A8 gene, we developed a novel LOVD database which includes clinically and genetically relevant data.

Materials and Methods

Subjects

In our diagnostic unit, a total of 1900 patients with a differential diagnosis of SLC6A8 deficiency were analyzed by DNA sequence analysis. This resulted in the detection of 66 individuals with intronic or synonymous variants (including 28 novel). These variants are addressed in the current study. Also, 5 novel patients affected with SLC6A8 deficiency are reported.

Methods

PCR of exon 1 to 13 of SLC6A8 (NM_005629.1)

Exon 2 to 13 and flanking intronic sequences of SLC6A8 were amplified using HotStarTaq Polymerase (Qiagen, USA). Amplification consisted of an initial denaturation step at 95°C for 15 minutes, followed by 38 cycles of 94°C for 45 seconds, 66°C for 45 seconds and 72°C for 80 seconds. For the amplification of exon 1 and its flanking sequences, Takara LA Taq (Takara Bio Inc, Japan) Polymerase was used. After an initial denaturing step for one minute at 94°C, amplification was allowed in 35 cycles composed of 95°C for 30 seconds, 66°C for 30 seconds and 72°C for 80 seconds.

RT-PCR analysis of SLC6A8 cDNAs and minigenes

RNA was isolated from lymphoblasts or fibroblasts using the SV RNA kit (Promega) or from PAX blood tubes (Qiagen). cDNA was synthesized from isolated RNA using Omniscript Reverse Transcriptase (Qiagen, USA) according to the manufacturer's instructions. RT-PCR was performed using cDNA primers designed for the exons of interest of the SLC6A8 gene. To rule out possible amplification of genomic DNA, RT-PCR for each RNA was also performed without reverse transcriptase.

Construction and transfection of the minigenes

Due to the large size of the SLC6A8 gene and its highly GC-rich 5'region, we only cloned the region of interest of the SLC6A8 gene. As templates for the PCR, the patients' and a wild type genomic DNAs were used. The fragment, covering exons 3 to 7 including 53 and 55 nucleotides of the flanking 5'and 3' intronic regions respectively, was amplified with forward primer 5'-CCGGAATTCGTAAAACGACGGCCAGCAGGGGGAGGTGGCCAGGG -3' containing an EcoR1 site and reverse primer 5'-GCGTCGACCAGGAAACAGCTATGACATGCATCTGGGTAGCACTC -3' with a SalI restriction site for cloning into the pBABE-puro plasmid. The fragment was amplified using Takara LA Taq (Takara Bio Inc, Japan), cloned into the TOPO-TA (Invitrogen, Breda, The Netherlands) vector and either wild type or mutant sequence was confirmed by sequencing. For the construction of the positive control (i.e. c.777+2T>A), site directed mutagenesis was performed on the TOPO vector containing the wild type fragment with forward primer 5'-AAATCCACGGGAAAGGAACCACTAGAGGCATGC-3' and reverse 5'-GCATGCCTCTAGTGGTTCCTTTCCCGTGGATTT-3'. Presence of the desired mutation and absence of PCR-artifacts were confirmed by sequencing of the complete fragments. After digestion with EcoR1 and Sal1, the inserts were sub cloned into the pBABE-puro plasmid.

Minigene constructs were transiently transfected into SLC6A8 deficient primary fibroblasts. With the use of polyethylenimine (PEI), 25 µg of construct, in a ratio of 3:1 (PEI:DNA), was transfected in fibroblasts grown to 70% confluence in a 75 cm 2 culture disc. After 48 hours, cells were harvested.

DNA sequence analysis

Sequence analysis was performed using BigDye v3.1 terminator and an ABI 3110XL (Applied Biosystems, The Netherlands). The obtained sequences were analyzed using the Mutation Surveyor software package (Softgenetics, State College, PA, USA).

Analysis of amplicons with DHPLC

For DHPLC analysis, optimal heteroduplex formation was realized by denaturing PCR products at 95°C for 5 min and gradual cooling to 25°C over a period of 60 min. Subsequently, 5 μl of the heteroduplex/homoduplex mixture was loaded on a WAVE 3500HT DNA fragment analysis system (Transgenomic, Omaha, NE). Elution of the PS-DVB DNAsep column (Transgenomic, Omaha, NE) was performed in highthroughput mode with a linear gradient of increasing acetonitril (ACN) concentration with a runtime of 3 min and a constant flow rate of 1.5 ml/min. Gradient was realized with the use of buffer A (0.1 M triethylammonium acetate [TEAA] and 0.025% ACN) and buffer B (0.1 M TEAA and 25% ACN). Navigator 2.0 software (Transgenomic, Omaha, NE) was used to calculate the ratio of buffer A and B during the gradient, as well as the optimal partially denaturing temperature (Tm) for the amplicon. The Tm of this amplicon was determined as 64°C.

LOVD Database

The target of the LOVD database is to include all published and unpublished mutations and variants. It is localized at a server in Leiden, The Netherlands, which can be reached by the following URL http://www.LOVD.nl/SLC6A8 or through the Variation Databases page of the Human Genome Variation Society (HGVS, www.HGVS.org/). The SLC6A8-specific database has been developed with the recently described LOVD software, [START_REF] Fokkema | LOVD: easy creation of a locus-specific sequence variation database using an "LSDB-in-a-box" approach[END_REF] which is in agreement with the HGVS guidelines. [START_REF] Dunnen | Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion[END_REF] All variants are checked and approved by Mutalyzer. [START_REF] Wildeman | Improving sequence variant descriptions in mutation databases and literature using the Mutalyzer sequence variation nomenclature checker[END_REF] So far 38 pathogenic mutations have been reported in 44 male patients affected with SLC6A8 deficiency.

Results

Validation of splice-site analysis using previously reported variants

We analyzed 7 pathogenic mutations (Table 1) detected in patients affected with SLC6A8 deficiency by applying five splice-site analysis tools (see methods). In 6 out of 7 pathogenic mutations at least four of the five tools predicted a reduced recognition of the splice sites, varying with a reduction of the probability score between 9% and 100%.

Three mutations had significant reduction scores of more than 99% in four of the five tools, whereas 3 other variants had reduction of their scores in a much lower range. One mutation, c.1392+24_1393-30del, reducing the intron from 76 to 52 bp, was not recognized by all the tools. Even though analysis of this mutation with Splice Predictor resulted in the loss of both rho and gamma values, this was not ascribed to the mutation itself but to Splice Predictor's precondition of an intron size of at least 60 bp.

In addition, 18 previously reported variants that are not disease causing were correctly predicted not to cause erroneous splicing by all five splice-site analysis tools, with the exception of one synonymous variant that showed in four tools minor reduction scores of equal or less than 7%. These variants are all confirmed to be non disease causing, since we now either established at the cDNA level that no erroneous splicing occurred or that the variants were detected in healthy control males or were published in dbSNP (See table 1; See www.lovd.nl/slc6a8).

Detection and classification of novel variants

Within the last decade we have analyzed about 1900 individuals (referred to our department because of e.g. mental retardation, increased urinary creatine/creatinine) by DNA sequence analysis of the SLC6A8 gene to exclude/confirm creatine transporter defect. In this endeavor we identified 41 variants including 28 novel synonymous and intronic variants that were not included in the dbSNP database and of which their (non-) disease causing nature needed to be established. All were subjected to the five splicesite analysis tools. In total, 3 novel pathogenic mutations (c.263-1G>C, c.778-2A>G, c.1596+1G>A) were detected that were predicted to cause aberrant splicing with reduction scores of 100% by at least three splice-site analysis tools (Table 2). Twentyseven variants showed no effect by all five tools. By only one or two tools 8 variants were predicted to decrease the possibility of correct splicing by 10% or less. One synonymous unclassified variant (c.780C>T) showed in three splice-site analysis tools reduction scores that appear significant in terms of percentage, but if the normal range is taken into account these differences are considered minimal (e.g. the score by the FruitFly tool shows a decrease of 32% while the actual score drops from 0.22 to 0.15 with the range of low probability to high probability being 0 -1.00). All these variants were considered non disease causing. Only 1 intronic unclassified variant (c.777+4C>T) was predicted by all five splice-site analysis tools to reduce the probability of the canonical donor site of exon 4 (Table 2) by 10%, 2%, 23%, 10% and 15% compared to the canonical donor site score. cDNA was not available from this patient and thus this variant was studied by overexpression of a minigene (i.e. a genomic SLC6A8 segment containing the c.777+4C>T variant). Both overexpression of the wild type minigene as well as the mutant minigene in SLC6A8 deficient primary fibroblasts showed normal splicing as detected by RT-PCR and sequence analysis. This is in contrast to the positive control minigene that contained the c.777+2T>A mutation and resulted in skipping of exon 4 (Figure 1).

Five novel SLC6A8 deficient patients.

The first case (Patient I) was a young female (DOB 01/93) born at term following a normal pregnancy and delivery with normal weight, length and head circumference.

From the age of 2 she developed a behavior disturbance and was assessed by a pedopsychiatric institution that also diagnosed mild mental retardation. At age 14 she was affected with auditory hallucinations and the diagnosis of chronic hallucinatory psychosis was made. Her cognitive impairment was stable with conserved language capacities and social contact, no motor impairment and no specific neurological signs.

Her total IQ was tested at 45 (verbal: 61, reason: 45, memory: 53; working velocity: 66). A brain H-MRS was performed which showed a reduced cerebral creatine level with a normal appearance to choline and N-acetylaspartic acid (Figure 2). Urinary creatine levels were within the upper normal range. Plasma creatine was determined twice resulting in values of 39.9 µmol/L and 75.4 µmol/L (normal 30 -124 µmol/L).

Genomic DNA sequencing revealed a c.263-1G>C mutation. This mutation arose de novo. Somatic mosaicism was not detected in her parents' DNA by both DNA analysis and the use of DHPLC (Figure 3). mRNA isolated from PAX tubes, followed by cDNA synthesis showed approximately 90% r.263_325del (p.Gly88_Leu108del) and 10% wildtype product. The X-inactivation was studied in blood with the analysis of HhaI digested and undigested DNA followed by PCR of the highly polymorphic CAG repeat of the androgen receptor gene and showed a 90:10 pattern. Also, fibroblasts incubated with a physiological concentration of creatine (25µM) showed no uptake. All these findings combined confirmed creatine transporter deficiency.

The second case (Patient II) was a 7 year old male with epilepsy, expressive language difficulties and a movement disorder. He was tested for mutations in SLC6A8 after H-MRS of the brain revealed a diminished creatine peak with a normal appearance to NAA and choline and urinary analysis showed an increased Cr/Crn value. This resulted in the detection of a hemizygous c.778-2A>G variant. His brother (Patient III), currently 10 years old, showed a similar clinical phenotype with an increased urinary Cr/Crn value and absence of cerebral creatine. He was also found to be hemizygous for this variant while his mother was proven to be heterozygous.

The fourth patient (Patient IV) was a young male with a reduced cerebral creatine level measured by H-MRS. Further investigation resulted in the detection of a c.1596+1G>A variant, which was also found to be heterozygous in DNA from his mother.

The fifth patient (Patient V) was unmistakably diagnosed with SLC6A8 deficiency at the age of 6 years. The patient suffered from moderate mental retardation and severe language delay. Quantitative localized single voxel magnetic resonance spectroscopy was performed over the basal ganglia and revealed a markedly diminished creatine level with a normal appearance to choline. Urinary analysis revealed a creatine/creatinine value of 1.7 (normal 0.017 -0.72). [START_REF] Almeida | Creatine and guanidinoacetate: diagnostic markers for inborn errors in creatine biosynthesis and transport[END_REF] The creatine uptake ability of the patients' fibroblasts was significantly decreased in comparison with control cells when incubated at a physiological creatine concentration of 25µM. Moreover, the mutation could not be detected in the DNA of the mother and is therefore considered de novo. At the cDNA level, two erroneous transcripts were revealed (r.[1392_1393ins1392+1_1393-1;1392+24_1393-30del,1393_1495del])(Figure 4). This conclusively classified the c.1392+24_1393-30del mutation as the pathogenic event. Also, the same de novo mutation was detected in an unrelated patient. [START_REF] Hathaway | X-Linked Creatine Transporter Deficiency Presenting as a Mitochondrial Disorder[END_REF]

LOVD database

In total, the LOVD database of SLC6A8 lists 44 SLC6A8 deficient families, with a total of 43 mutations. Of these, 38 are proven pathogenic (+/+), whereas 5 others are presumed to be pathogenic (+?/+?). The latter are all missense variants, which have not been investigated by overexpression studies yet. [START_REF] Rosenberg | Functional characterization of missense variants in the creatine transporter gene (SLC6A8): improved diagnostic application[END_REF] The database (Figure 5) provides detailed information on the nature of the variants, but also exon/intron location of the DNA change (both according to current nomenclature and as published), RNA change and protein change. To all variants a unique database number is assigned. The column "variant remarks" provides a detailed description on the nature of the variant. The final conclusion is presented in the first column "path."

where the first sign provides the conclusion of the report and the second one that of the curator (e.g. +/+ meaning confirmed pathogenic). Other columns include detailed information on the variant origin, reference, template, technique, frequency, disease status, patient remarks, times reported, gender, geographic origin and ethnic origin.

Moreover, clinical symptoms are continuously archived (non-public). The database is an excellent tool to get an up-to-date overview of the type of mutations, the frequency of specific variants and more details. This data can be found in the different tabs by any user, but an advanced search function is also available. Researchers or clinicians who are interested in the nature of a specific variant can use the database to search if the variant has been detected in other patients. This information may have been published in reports or on the website only, but also may include unpublished variants which have not yet been made public. The latter allows contact between clinicians even if the data is not publicly released. All scientists/clinicians are invited to submit their data to the database. The identity of the submitter is included in the database.

Discussion

In this study, the nature of 67 intronic and synonymous variants have been studied by free web based splice-site analysis tools. These have been characterized and are all being included in a newly developed LOVD database. In addition, all previously published mutations are included in this database as well. This is of high relevance for researchers and clinicians who detect difficult to interpret variants in the SLC6A8 gene.

In an ideal diagnostic setup, an unclassified variant is further investigated at the mRNA, protein and/or functional level to confirm/exclude its pathogenic nature. However, in diagnostic laboratories time is usually limited and proper materials are often not available. Therefore, alternative methods are warranted for variant classification such as frequently used splice-site analysis tools, which predict the possible effect on RNA splicing of a variant based on known and computed conserved splicing sequences. Here we demonstrate that the use of five splice-site analysis tools, indeed is very helpful for proper classification.

Twenty four out of 25 (96%) variants were properly classified by the combined use of the five tools. The importance of using more than one splice-site analysis tool is illustrated by the fact that 4 out of 7 proven pathogenic mutations were not predicted to have a strong effect (i.e. 90% or higher reduction) by one or more of the individual analysis tools, whereas the combined data of all the tools clearly predicted erroneous splicing for 6 of these variants (Table 1). In one case, only one tool predicted a reduction in the probability of correct splicing. The fact that this "missed" mutation (c.1392+24_1393-30del) comprises an intronic deletion of 24 base pairs out of a 76 base pairs intron, easily explains this omission as none of the five splice-site analysis tools have intron size as a parameter. This caveat is an important finding for diagnostic laboratories that encounter this specific type of mutations (e.g. deletions, insertions in small introns). We demonstrated that this deletion indeed results in erroneous spliced transcripts (r.[1392_1393ins1392+1_1393-1;1392+24_1393-30del,1393_1495del]), (Abdul-Rahman et al, in press, present study).

In 17 out of 18 proven variants, no change in prediction scores was detected by any of the five tools. Only 1 variant, that was proven not to interfere with proper splicing, had a reduced score of 7% or less compared to the canonical site by four of the five tools.

Due to this variant, we arbitrarily decided to only consider variants for further molecular workup if at least three tools predicted a mild effect (10 < > 80%), or if one or two tools predicted a significant effect (>80%). In addition, we took into account the prediction score of the wild type splice site. Thus a 50% reduction of a canonical site with a 0.99 score is more likely to have an effect on splicing than a 50% reduction in a splice site with a much weaker score of for instance 0.22.

Utilizing these criteria we analyzed the potential effect of 41 unclassified variants on the SLC6A8 mRNA with the use of these five splice-site analysis tools. This resulted in the identification of 3 pathogenic mutations (c.263-1G>C, c.778-2A>G, c.1596+1G>A), and 38 variants that have no apparent relation with creatine transporter deficiency. The effect on splicing for 2 of the 3 novel pathogenic mutations could not be confirmed at the cDNA level. However, given the fact that all 3 mutations affect the donor or acceptor splice-site, all were classified as pathogenic. Pathogenicity was in excellent agreement with the fact that the mutations were not detected in 280 control chromosomes, the biochemical findings (increased urinary Cr/Crn) and/or reduced cerebral creatine detection by H-MRS.

Interestingly, one novel mutation (c.263-1G>C) was found in a girl, who according to our knowledge is the first reported index girl affected with SLC6A8 deficiency. The girl was not correctly diagnosed until the age of 14 when a brain H-MRS revealed a reduced cerebral creatine level. This led to DNA analysis which resulted in the discovery of the pathogenic c.263-1G>C mutation. Additional investigations revealed a urinary creatine to creatinine ratio within the normal range as well as normal values for plasma creatine.

The discrepancy between the urinary, plasma and cerebral creatine levels is possibly caused by variation of the X-inactivation pattern in different tissues, which was shown to be skewed in blood of this patient.

In a recently described cohort of female relatives who were heterozygous for a pathogenic mutation in SLC6A8, it was shown that symptoms of SLC6A8 deficiency (e.g. mental retardation, learning difficulties and constipation) can occur in female heterozygotes. However, it should be noted that the level of biochemical markers (urinary creatine to creatinine ratio and/or cerebral creatine) usually overlap with that of normal controls. This is in concordance with the clinical and biochemical findings of the above mentioned female index patient and confirms the recommendation of screening for mutations in SLC6A8 in females with (mild) mental retardation/psychiatric disorders as this method appears to be the most sensitive and specific test. [START_REF] Van De Kamp | Clinical features and X-inactivation in females heterozygous for creatine transporter defect[END_REF] In only 1 of the 38 UVs, mild reduction values of the probability scores were detected by all five splice-site analysis tools. Unfortunately it was not possible to obtain additional material of the patient to isolate mRNA, and clinically it was also not possible to exclude/confirm SLC6A8 deficiency. We therefore selected this variant (c.777+4C>T) for overexpression studies of a minigene containing the variant. These studies revealed no aberrant splicing and therefore this variant is also considered nonpathogenic.

After evaluating the results of the splice-site analysis tools reported in this study, we defined our criteria for the analysis of SLC6A8. If at least three out of five splice-site analysis tools predict a reduction of more than 10% or one out of five predicts a reduction of 80% of the site score compared to the canonical site, further research is warranted. However a critical view needs to be kept which is illustrated by the fact that in our study 1 out of the 10 proven pathogenic mutations would have escaped proper identification. The c.1392+24_1393-30del mutation was not recognized by the overall prediction score of all five splice-site analysis tools. It is of note that these scores do not include composition, size and branch site of the intron as parameters. One exception is Splice predictor, which does have additional intron dependent scores, namely rho and gamma. Unfortunately, these scores are not calculated and set at zero when an intron is smaller than 60 base pairs, therefore making it difficult to deduce the actual reduction with this variant as the deletion leads to a 52 base pair intron. This of course reduces the reliability of these scores since naturally occurring introns can have sizes of 60 base pairs and less.

In conclusion, splice-site analysis tools are very important in the process of classifying novel variants. For definite classification of novel variants outside the canonical donor and acceptor sites, in vitro experimentation, either with mRNA analysis or with the use of a minigene (if no additional material is available), is an essential procedure.

Titles and Legends to Tables and Figures

Table 1

Proven pathogenic mutations and non disease associated variants in SLC6A8.

Validation of the splice-site analysis tools.

a This mutation arose de novo.

Table 2

Twenty eight novel intronic variants and 13 previously reported unclassified intronic variants in SLC6A8.

None of these variants, including 3 pathogenic mutations and 13 previously reported variants were detected in 280 control alleles.

Location refers to the intron (i) or exon (2 digits) number in which the variant was found. r.spl?: the variant is expected to affect splicing; however, no further mRNA analysis is performed.

a This variant was cloned into a minigene. Overexpression studies indicated that this variant does not affect splicing. NC: no change (i.e. the variant did not influence the predicted score compared to the canonical site) SND: site not detected. The canonical donor or acceptor site is not recognized as a naturally occurring site by this splice-site analysis tool.

Path.: Reported / concluded pathogenicity. -: No known pathogenicity; -?: Probably no pathogenicity; ?: Unknown; +?: Probably pathogenic; +: Pathogenic.

Deduced effect: Indicates that the variant has not been tested at the mRNA level; r.(spl?): the variant is expected to affect splicing; p.(=), no effect predicted, but not tested at the mRNA level. A proton MRS was performed on a 14 year old girl with mild mental retardation. Creatine (Cr) was found to be reduced with a normal appearance to choline (Cho) and The c.1392+24_1393-30del variant was found in two independent patients and occurred de novo in both cases. In the patients' DNA a hemizygous variant, c.1392+24_1393-30del, was found (upper pane). mRNA analysis showed that this variant affects splicing and results in different splice products, r.[1392_1393ins1392+1_1393-1;1392+24_1393-30del, 1393_1495del](lower pane), resulting in SLC6A8 deficiency.

For the second patient see Hathaway et al. [START_REF] Hathaway | X-Linked Creatine Transporter Deficiency Presenting as a Mitochondrial Disorder[END_REF] This variant is annotated according to the guidelines of Den Dunnen and Antonarakis (http://www.genomic.unimelb.edu.au/mdi/mutnomen/).

Figure 5

Screenshot of the newly developed LOVD/SLC6A8 database.

The currently known variants and pathogenic mutations of SLC6A8 are described in the newly developed LOVD database including the corresponding clinical data. Search queries can be performed for many parameters (i.e. specific variant, exon number, biochemical findings). In order to submit variants it is required to log in, after which the variants will be checked for correctness and completeness by the curator.

  b r.[1392_1393ins1392+1_1393-1;1392+24_1393-30del, 1393_1495del] Location refers to the intron (i) or exon (2 digits) number in which the variant was found. In the upper panel the proven pathogenic variants are denoted. The lower panel includes both intronic and synonymous variants. NC: no change. Variant did not influence the predicted score compared to the canonical site. Path.: Reported and concluded pathogenicity. +: Pathogenic; -: No known pathogenicity. r.spl?: the variant is expected to affect splicing; however, no further mRNA analysis is performed. Ref: Reference. Ref 2: 2 ; Ref 4: 4 ; Ref 17: 17 ; Ref 18: 18 ; Ref 19: 19 ; Ref 20: 20 ; Ref 21: 21 . cDNA denotation is based on the reference sequence NM_005629.3, where +1 corresponds to the first nucleotide of the initiation codon. All variants are annotated according to the guidelines of Den Dunnen and Antonarakis (http://www.genomic.unimelb.edu.au/mdi/mutnomen/).

  Ref: Reference. Ref 2: 2 ; Ref 4: 4 . cDNA denotation is based on the reference sequence NM_005629.3, where +1 corresponds to the first nucleotide of the initiation codon. All variants are annotated according to the guidelines of Den Dunnen and Antonarakis (http://www.genomic.unimelb.edu.au/mdi/mutnomen/).

Figure 1 RT

 1 Figure 1

Figure 2 Proton

 2 Figure 2

N

  -acetylaspartic acid (NAA). Subsequent genomic DNA sequencing revealed a pathogenic c.263-1G>C mutation, which confirmed the diagnosis of SLC6A8 deficiency.

Figure 3 Patient

 3 Figure 3

Figure 4

 4 Figure 4

Table 1 .

 1 Proven pathogenic mutations and non disease associated variants in SLC6A8 . Validation of the splice-site analysis tools.

	Location cDNA	RNA	Protein	Controls	Netgene 2	Fruitfly	Splice Predictor	Genscan W	FSplice	Path.	Reference
	01i	c.262_262+1delinsTT a	r.(spl?)	p.(?)	0/280	1.00 > 0 (-100%) 0.99 > 0.01 (-99%)	0.99 > 0 (-100%)	57.0 > 39.1 (-31%) 12.4 > 0 (-100%) +/+
	01i	c.263-2A>G	r.263_328del	p.Gly88_Glu109del	0/280	0.95 > 0 (-100%) 0.61 > 0 (-100%)	0.97 > 0 (-100%)	21.9 > 5.9 (-73%) 12.5 > 0 (-100%) +/+
	05	c.912G>A	r.772_912del	p.Ile260_Gln304del	0/280	0.99 > 0.80 (-19%) 0.95 > 0.67 (-30%)	0.99 > 0.88 (-11%)	12.4 > 9.1 (-27%) 11.6 > 6.0 (-48%) +/+
	06i	c.1016+2T>C	r.(spl?)	p.(?)	0/280	0.99 > 0 (-100%) 0.99 > 0 (-100%)	0.97 > 0 (-100%)	18.2 > 10.9 (-40%) 10.16 > 0 (-100%) +/+
	07	c.1141G>C	r.[1141G>C, 1129_1141del] p.[Gly381Arg, Val377GlyfsX15] 0/280	0.99 > 0.90 (-9%) 0.92 > 0.17 (-82%)	0.99 > 0.90 (-10%)	24.3 > 18.7 (-23%) 11.0 > 1.5 (-86%) +/+
	09i	c.1392+24_1393-30del r.[1392...] b	p.(?)	0/280	NC	NC	NC	NC	NC	+/+
	10i	c.1495+5G>C	r.1393_1495del	p.Gly465GlufsX12	0/280	0.95 > 0.70 (-26%) 0.96 > 0 (-100%)	0.99 > 0.86 (-13%)	17.9 > 15.7 (-12%) 11.0 > 1.5 (-86%) +/+
	01i	c.262+26T>C	ND	p.(=)	6/280	NC	NC	NC	NC	NC	-/-
	02i	c.394+88G>C	ND	p.(=)	rs6643763, 7/280 NC	NC	NC	NC	NC	-/-
	05	c.813C>T	ND	p.=	4/280	NC	NC	NC	NC	NC	-/-
	06i	c.1016+9C>T	r.=	p.=	0/280	NC	NC	NC	NC	NC	-/-
	07i	c.1141+37G>A	r.=	p.=	rs2071028, 9/280 NC	NC	NC	NC	NC	-/-
	07i	c.1141+87A>G	r.=	p.=	rs41302172, 6/280 NC	NC	NC	NC	NC	-/-
	07i	c.1142-152_151del	r.=	p.=	0/280	NC	NC	NC	NC	NC	-/-
	07i	c.1142-35G>A	r.=	p.=	2/280	NC	NC	NC	NC	NC	-/-
	08i	c.1254+28C>T	r.=	p.=	0/280	NC	NC	NC	NC	NC	-/-
	08i	c.1255-35G>A	r.=	p.=	0/280	NC	NC	NC	NC	NC	-/-
	09i	c.1393-36G>A	r.=	p.=	1/280	NC	NC	NC	NC	NC	-/-
	10	c.1494C>T	r.=	p.=	1/280	0.95 > 0.91 (-4%) 0.96 > 0.89 (-7%)	NC	17.9 > 16.7 (-7%) 11.0 > 10.3 (-6%) -/-
	10i	c.1496-18C>T	r.=	p.=	0/280	NC	NC	NC	NC	NC	-/-
	11i	c.1596+21G>A	r.=	p.=	0/280	NC	NC	NC	NC	NC	-/-
	12	c.1678A>G	ND	p.=	1/280	NC	NC	NC	NC	NC	-/-	2.4
	12i	c.1767+15C>T	r.=	p.=	0/280	NC	NC	NC	NC	NC	-/-
	12i	c.1767+32C>A	r.=	p.=	0/280	NC	NC	NC	NC	NC	-/-
	12i	c.1768-82G>C	ND	p.(=)	1/280	NC	NC	NC	NC	NC	-/-

Table 2 .

 2 Twenty eight novel intronic variants and 13 previously reported unclassified intronic variants in SLC6A8.

	Location cDNA		Deduced effect Netgene 2	Fruitfly	Splice Predictor	Genscan W	FSplice	Path.	Ref
	01i	c.263-1G>C $	r.263_325del	0.95 > 0 (-100%)	0.93 > 0.32 (-66%) 0.97 > 0 (-100%) 21.9 > 5.9 (-73%) 12.5 > 0 (-100%) +/+
	01i	c.262+65G>A	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	02	c.306A>G		p.(=)?	NC	NC	NC	NC	NC	-?/-?
	02i	c.394+52C>T	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	02i	c.394+66C>A	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	02i	c.394+72C>A	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	02i	c.394+88G>A	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	03	c.495C>T		p.(=)?	NC	NC	NC	33.3 > 32.8 (-2%) NC	-?/-?
	03	c.603C>T		p.(=)?	NC	NC	NC	NC	NC	-?/-?
	03i	c.644+9G>A	p.(=)?	NC	NC	0.85 > 0.79 (-7%) NC	NC	-?/-?
	04i	c.778-2A>G		r.(spl)?	1.00 > 0 (-100%)	0.22 > 0 (-100%)	SND	12.4 > 7.7 (-38%) 3.3 > 0 (-100%)	+/+
	04i	c.777+4C>T	a	p.(=)?	0.91 > 0.82 (-10%) 0.61 > 0.47 (-23%) 0.94 > 0.92 (-2%) 7.8 > 6.6 (-15%)	9.6 > 8.7 (-10%)	-/-
	05	c.780C>T		p.(=)?	1.00 > 0.96 (-4%) 0.22 > 0.15 (-32%) NC	NC	3.3 > 2.67 (-20%) -?/-?
	05	c.856C>T		p.(=)?	NC	NC	NC	NC	NC	-/-
	05i	c.913-40T>C	p.(=)?	NC	NC	NC	NC	10.0 > 9.2 (-9%)	-?/-?
	06i	c.1016+41_46del	p.(=)?	0.99 > 0.96 (-3%) NC	NC	NC	NC	-?/-?
	06i	c.1016+41_45dup	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	06i	c.1017-38C>G	p.(=)?	NC	NC	NC	NC	10.2 > 10.0 (-2%) -?/-?
	07i	c.1141+18G>A	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	07i	c.1142-130C>T	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	07i	c.1142-122C>T	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	07i	c.1142-100G>A	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	07i	c.1142-98C>A	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	07i	c.1142-19G>A	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	08	c.1162G>A		p.(=)?	NC	NC	NC	NC	NC	-?/-?
	08i	c.1254+39G>T	p.(=)?	NC	NC	0.92 > 0.89 (-3%) NC	NC	-?/-?
	08i	c.1254+54C>T	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	09i	c.1392+12G>T	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	09i	c.1392+31T>C	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	10	c.1416G>A		p.(=)?	NC	NC	NC	NC	NC	-/-
	10	c.1437C>T		p.(=)?	NC	NC	NC	NC	NC	-?/-?
	10i	c.1496-8C>T	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	10i	c.1496-7G>A	p.(=)?	1.00 > 0.96 (-4%) 0.72 > 0.67 (-7%) NC	NC	NC	-?/-?
	10i	c.1496-5C>T	p.(=)?	NC	NC	0.86 > 0.81 (-6%) NC	NC	-?/-?
	11i	c.1596+1G>A	r.(spl)?	0.95 > 0 (-100%)	1.00 > 0 (-100%)	0.99 > 0 (-100%) 14.19 > 0 (-100%) 11.98 > 0 (-100%) +/+
	11i	c.1596+24_1597-41dup p.(=)?	NC	NC	NC	NC	NC	-?/-?
	11i	c.1596+28C>T	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	11i	c.1597-20C>T	p.(=)?	NC	NC	NC	NC	NC	-?/-?
	12	c.1629G>A		p.(=)?	NC	NC	0.57 > 0.55 (-4%) 27.9 > 25.3 (-9%) NC	-?/-?
	12	c.1662G>A		p.(=)?	NC	NC	NC	NC	NC	-?/-?
	12i	c.1767+20C>T	p.(=)?	NC	NC	NC	NC	NC	-?/-?

Acknowledgments

The authors thank Donald E Brass for his excellent reviewing. The work of Ofir T Betsalel, Efraim H Rosenberg, Ligia S Almeida and Gajja S Salomons is supported by the Dutch society for Scientific Research (ZonMW/NWO), VIDI grant number 917.56.349 and the Horst Bickel Award 2002, that of Tjitske Kleefstra by the European Union grant QLG3-CT-2002-01819 EURO-MRX and that of Charles Schwartz by grants of NICHD (HD26202) and the South Carolina Department of Disabilities and Special Needs.

In silico analysis

The analysis of splicing efficiencies in the normal and mutant sequences was carried out using the following five splice-site analysis tools which are commonly used in diagnostic laboratories: the Berkeley Drosophila Genome Project 6 (BDGP, http://www.fruitfly.org/seq_tools/splice.html), Netgene2 [START_REF] Hebsgaard | Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information[END_REF] (http://www.cbs.dtu.dk/services/NetGene2/), Splice Predictor 8 (http://deepc2.psi.iastate.edu/cgi-bin/sp.cgi), GenscanW 9 (http://genes.mit.edu/GENSCAN.html) and FSplice (http://linux1.softberry.com/berry.phtml?topic=fsplice&group=programs&subgroup=gfi nd). The parameters used for analysis were the default settings of all tools.


Manish Siddharth

Elena Banka

Stavit Alon Chervinsky

William Shalev

Dian Newman

Professor Yanick Donnai

Shay Crow

Joanne Yeganeh

Yacobovich

Dr Siddharth Banka
email: siddharth.banka@manchester.ac.uk

Elena Chervinsky

William G Newman

Yanick J Crow

Shay Yeganeh

Joanne Yacobovich

Dian Donnai

Stavit Shalev

Further delineation of the phenotype of severe congenital neutropenia type 4 due to mutations in G6PC3

Keywords:

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Abstract

Severe congenital neutropenia type 4 (SCN4) is an autosomal recessive condition which was defined recently with identification of the causative mutations in G6PC3. To date there are only three reports in the literature describing patients with SCN4 with mutations in the G6PC3 gene. We report four individuals with SCN4 who belong to a single large consanguineous kindred. We provide an overview of the non-haematological features of the condition with a focus on the adult phenotype which has not been previously described in detail. We show that the superficial venous changes seen in SCN4 patients can develop into varicose veins and venous ulcers in adulthood. We review the range of congenital anomalies associated with SCN4. We demonstrate that secundum atrial septal defect, patent ductus arteriosus and valvular defects are the most frequent cardiac anomalies in SCN4.

Drawing parallels with type 1 glycogen storage disease, we propose that poor growth of prenatal onset, mild to moderate learning disability, primary pulmonary hypertension, delayed or incomplete puberty, hypothyroidism and dysmorphism likely represent features of this syndrome. We also suggest monitoring for lipid anomalies, and kidney and liver function in affected patients. Delineation of the SCN4 phenotype may help in appropriate treatment and management and provide further insights into the pathogenesis of this multisystem disease.

Introduction

The congenital neutropenias are a group of heterogeneous disorders. To date, seventeen causative genes have been identified for syndromic and non-syndromic causes [START_REF] Klein | Molecular basis of congenital neutropenia[END_REF] . Severe congenital neutropenia type 4 (SCN4, OMIM 612541) was recently defined with identification of the causative mutations in G6PC3 (Gene ID 92579) and to date there are only three reports in the literature describing fifteen patients in total [START_REF] Arostegui | A novel G6PC3 homozygous 1-bp deletion as a cause of severe congenital neutropenia[END_REF][START_REF] Boztug | A Syndrome with Congenital Neutropenia and Mutations in G6PC3[END_REF][START_REF] Xia | Prevalence of mutations in ELANE, GFI1, HAX1, SBDS, WAS and G6PC3 in patients with severe congenital neutropenia[END_REF] . SCN4 has a distinct phenotype showing the presence of a prominent superficial venous pattern and an increased susceptibility to infection. Other described features include intermittent thrombocytopenia, hepatosplenomegaly, congenital heart defects, urogenital malformations, cleft palate, inguinal hernia, microcephaly, sensorineural hearing loss and myopathy [START_REF] Boztug | A Syndrome with Congenital Neutropenia and Mutations in G6PC3[END_REF] .

We report four individuals from a single large consanguineous Israeli kindred of Arab-Muslim origin with severe congenital neutropenia, a prominent superficial venous pattern and additional clinical features. The pedigree is shown in Figure 1 and the clinical details are provided below.

Clinical description

Patient 1 (V-7) is a 29 year old female born to first cousin parents. She was small for gestation at birth. Her adult height is on the 2 nd centile and her weight and occipito-frontal circumference (OFC) are on the 5 th centile. She also has mild learning disability and required special education. She has irregular menses. She has suffered from recurrent pneumonia, otitis media and perianal abscesses. Significant results of her haematological and biochemical investigations are summarized in table 1. On examination she has a prominent superficial venous pattern and varicose veins. She also shows mild kyphosis, tapering fingers and clinodactyly of the 4 th and 5 th fingers.

Patient 2 (V-13) is a 26 year old man. He is IV-7's second cousin on the maternal side and third cousin on the paternal side. At birth he was small for dates and failed to thrive in infancy. His current height is below the 0.4 th centile and weight is on the 2 nd centile.

Agenesis of the left kidney and right hydronephrosis were noted in childhood. His puberty was delayed. He also has mild learning disability and went to a special school. He has experienced recurrent episodes of pneumonia and otitis media. His relevant haematological and biochemical investigation results are presented in table 1. On examination he has a prominent superficial venous pattern and varicose veins which have resulted in a venous stasis ulcer. He has poorly developed secondary sexual characteristics and gynaecomastia.

He has full cheeks (Figure 2a) and fat deposition around his trunk. His anterior and posterior hairlines are low (Figure 2b) and he has broad thumbs.

One of his younger brothers (V-10) died soon after birth due to a congenital heart defect, about which more details are not available. He has an older sister (V-14) who has learning difficulties but does not share any other clinical features of SCN4.

Patient 3 (V-17) was V-13's double first cousin. Patent ductus arteriosus (PDA) and secundum atrial septal defect (ASD) were noted at birth. Her adult height, weight and OFC were all on the 2 nd centile. Menarche was not delayed but secondary sexual characteristics were slow to develop. She also had mild learning difficulties and went to a special-needs school. She suffered from recurrent urinary tract infections and herpes stomatitis. She died at the age of 25 years due to sepsis. Her peripheral blood counts and bone-marrow aspiration results are summarized in table 1. On examination she had prominent and widespread superficial venous pattern and varicose veins on the lower legs. She had acne on her face and trunk. Her breasts and pubic hair were between Tanner stages 3 and 4 in adulthood. She had bilateral keratoconus.

Patient 4 (V-21) is a two old year boy born to first cousin parents. His parents belong to the same kindred of the first three patients described above, although the exact relationship is not known. He was diagnosed prenatally to have pulmonary valve stenosis. At eight days of age he underwent pulmonic balloon valvuloplasty. PDA and secundum ASD were also noted. Soon after birth he developed pulmonary hypertension of unknown cause, which was treated with sildenafil until eight months of age. He had bilateral undescended testis and gastro-oesophageal reflux. He was also found to have raised gamma-glutamyl transferase and calcifications in the liver at seven days of age. At age three months he was diagnosed with choledocholithiasis and was treated with ursodeoxycholic acid. He is still failing to thrive and has global mild to moderate developmental delay. His current length is on the 5 th centile, weight below 0.4 th centile and OFC on the 0.4 th centile. He suffers with recurrent otitis media. His pre-treatment peripheral blood counts and bone-marrow aspiration results are provided in table 1. On examination he has plagiocephaly, sparse scalp hair, mid-face hypoplasia and a tented mouth. He also has a prominent superficial venous pattern on his head, a pectus carinatum and a small umbilical hernia. He has loose skin on the palms and soles and an uneven fat distribution.

Materials and Methods

Ethical approval for this study was obtained from the University of Manchester (06138) and NHS ethics committee (06/Q1406/52). DNA was extracted from lymphocytes from the four affected individuals using standard protocol. All six exons of G6PC3 were amplified by polymerase chain reaction in the first three patients using the primers described by Boztug et al. In patient four, only exon six of G6PC3 was amplified. V-14 has not been tested for the familial mutation. Genomic DNA sequence was analyzed using ABI 3130 and Staden software.

Results

All four patients were homozygous for the c.758G>A mutation in G6PC3 (Figures 3a &b) which results in substitution of arginine to histidine at position 253 (p.R253H). This mutation has been described previously [START_REF] Boztug | A Syndrome with Congenital Neutropenia and Mutations in G6PC3[END_REF] . We were unable to establish if this mutation was on a common haplotype with previously reported cases or whether this residue represents a mutational hotspot. Previous cases with the same mutation have been of Turkish Arameanian background, which would be consistent with a shared Mediterranean ancestry with the Israeli Arabs [START_REF] Arnaiz-Villena | HLA alleles and haplotypes in the Turkish population: relatedness to Kurds, Armenians and other Mediterraneans[END_REF] .

Discussion

Glucose-6-phosphatase catalyzes the final step in glycogenolysis, i.e. the conversion of glucose-6-phosphate to glucose. This step takes place in the endoplasmic reticulum (ER).

Glucose-6-phosphatase is encoded by a gene-family consisting of G6PC1, G6PC2 and, G6PC3 [START_REF] Hutton | Glucose-6-phosphatase Catalytic Subunit Gene Family[END_REF] . G6PC1 is expressed in the liver, kidney and small intestine. Homozygous lossof-function mutations in G6PC1 cause type 1a glycogen storage disease (GSD1a) [START_REF] Lei | Mutations in the Glucose-6-Phosphatase Gene That Cause Glycogen Storage Disease Type 1a[END_REF] . GSD1a is characterized by fasting hypoglycemia, hepatomegaly, growth retardation, fasting lactic acidosis, hyperlipidemia and hyperuricemia. Long-term complications of GSD1a include hepatic adenomas, pancreatitis, gout, renal failure, pulmonary hypertension, polycystic ovaries, osteoporosis, platelet dysfunction, and mild to moderate learning difficulties [START_REF] Janecke | Molecular Genetics of Type 1 Glycogen Storage Disease[END_REF] .

The transport of glucose-6-phosphate from the cytosol to ER is facilitated by glucose-6phosphate translocase, encoded by the ubiquitously expressed SLC37A4 [START_REF] Veiga-Da-Cunha | The putative glucose 6-phosphate translocase gene is mutated in essentially all cases of glycogen storage disease type I non-a[END_REF] . Homozygous mutations in SLC37A4 result in GSD1b, which has a more severe phenotype than GSD1a and, of note, also causes chronic neutropenia.

G6PC3 is expressed ubiquitously and is important in maintaining neutrophil viability [START_REF] Boztug | A Syndrome with Congenital Neutropenia and Mutations in G6PC3[END_REF] .

Loss of G6PC3 function results in increased susceptibility to apoptosis in skin fibroblasts, neutrophils and myeloid cells. Increased apoptosis is likely due to decreased intra-cellular glucose concentrations and mediated via an ER stress mechanism and may predispose to associated congenital malformations.

Including the four patients presented here, a total of nineteen SCN4 cases have now been described in the literature [START_REF] Arostegui | A novel G6PC3 homozygous 1-bp deletion as a cause of severe congenital neutropenia[END_REF][START_REF] Boztug | A Syndrome with Congenital Neutropenia and Mutations in G6PC3[END_REF][START_REF] Xia | Prevalence of mutations in ELANE, GFI1, HAX1, SBDS, WAS and G6PC3 in patients with severe congenital neutropenia[END_REF] . Congenital neutropenia, frequent infections and prominent superficial veins are consistent features in these patients. We provide an overview of the non-haematological features of the condition with a focus on the adult phenotype which has not previously been described in detail. We draw parallels between the phenotype of SCN4 patients with GSD1a because both G6PC3 and G6PC1 code for the enzyme glucose-6phosphatase and with GSD1b because of the known functional relationship of the enzyme with glucose-6-phosphate transporter. A summary of major clinical features is provided in table 2.

Venous anomalies:

The prominent superficial venous pattern, which is the most distinctive non-haematological feature of SCN4, is less evident in infancy and gradually becomes more prominent with age. Considering the patients described here, it is clear that these vascular changes can develop into varicose veins in adulthood resulting in venous ulcers.

Patients with SCN4 should be monitored for this complication. This leads us to conclude that the pulmonary disease in this patient was primary. Of note, Dursun et al recently reported a pair of Turkish siblings, one boy and one girl, with primary pulmonary hypertension, leucopenia and secundum ASD [START_REF] Dursun | Familial pulmonary arterial hypertension, leucopenia, and atrial septal defect: a probable new familial syndrome with multisystem involvement[END_REF] . Both the siblings with Dursun syndrome (OMIM 613034) demonstrated a phenotype that is strikingly similar to that of the children described with SCN4. Primary pulmonary hypertension is also a well recognized feature of GSD1 [START_REF] Humbert | Severe pulmonary arterial hypertension in type 1 glycogen storage disease[END_REF] and may be an additional feature of SCN4.

Growth:

Poor growth of prenatal onset may also be a core feature of SCN4. In four affected individuals presented here showed intrauterine growth retardation and demonstrated a failure to thrive in infancy. Childhood stature and final adult height seem to be at the lower end of the normal range in all the four cases. The height and weight of the boy described by Arostegui et al were below the 3 rd percentile 2 and at least seven of the twelve patients reported by Boztug et al demonstrated growth retardation or height/weight at the lower end of the normal range [START_REF] Boztug | A Syndrome with Congenital Neutropenia and Mutations in G6PC3[END_REF] . We note that female g6pc3 -/-mice exhibit growth retardation [START_REF] Wang | Deletion of the Gene Encoding the Ubiquitously Expressed Glucose-6-phosphatase Catalytic Subunit-related Protein (UGRP)/Glucose-6-phosphatase Catalytic Subunit-beta Results in Lowered Plasma Cholesterol and Elevated Glucagon[END_REF] , but in humans this features seems not to be sex-limited.

Development: All four patients described here have mild to moderate developmental delay and/or learning disability. All three adult patients required special education and none have ever been in paid employment. Patient 2 has a sibling with learning disability who does not share the rest of the SCN4 phenotype and therefore the possibility of a different cause for learning difficulties in this kindred cannot be ruled out. The male reported by Arostegui et al also had learning difficulties, while no specific comments were made about neurodevelopmental aspects in the series described by Boztug et al and Jun et al. We suggest that the neuro-developmental aspect of the SCN4 phenotype warrants further investigation.

Endocrine dysfunction: Of fifteen previously reported patients, only two were beyond pubertal age -a 22 year old male described by Arostegui et al and a 17 year old male described by Boztug et al [START_REF] Arostegui | A novel G6PC3 homozygous 1-bp deletion as a cause of severe congenital neutropenia[END_REF][START_REF] Boztug | A Syndrome with Congenital Neutropenia and Mutations in G6PC3[END_REF] . The ages of the two patients described by Jun et al were not given [START_REF] Xia | Prevalence of mutations in ELANE, GFI1, HAX1, SBDS, WAS and G6PC3 in patients with severe congenital neutropenia[END_REF] . Three of the patients described here are over the age of 25 years, thus providing an insight into the adult SCN4 phenotype. Raised TSH was found in patients 1 and 2, suggesting that hypothyroidism may represent a feature of SCN4. Poor secondary sexual development was seen in two of our patients (patient 2 and 3) and we note that g6pc3 -/- mice exhibit significant reduction in g6p hydrolytic activity in the testes, although without any known phenotypic consequence. We note also that delayed puberty is a known feature of GSD1. We suggest that evaluation of pubertal development in adolescents and thyroid function assessment should be considered in the follow-up of SCN4 affected patients.

Lipid abnormalities: Patients with GSD1 often have dyslipidemia, which is thought to be due to increased de novo lipogenesis and delayed conversion of Very low density lipoproteins to intermediate lipoprotein particles [START_REF] Bandsma | Increased de novo Lipogenesis and Delayed Conversion of Large VLDL into Intermediate Density Lipoprotein Particles Contribute to Hyperlipidemia in Glycogen Storage Disease Type 1a[END_REF] . Patient 1, the oldest in our series, had low HDL levels. She also had persistently increased amylase activity. The risk of pancreatitis due to dyslipidemia is increased in GSD1 [START_REF] Rake | Glycogen storage disease type I: diagnosis, management, clinical course and outcome. Results of the European study on glycogen storage disease type I (ESGSD I)[END_REF] . Our findings suggest that lipid profile should be monitored in adults with SCN4.

Liver and kidney function: Findings of raised gamma-glutamyl transferase, liver calcifications and choledocholithiasis in patient 4, and elevated uric acid level in patient 1, indicates that liver function, kidney function and uric acid levels should be monitored in SCN4 similar to patients with GSD1.

Other congenital anomalies and minor dysmorphism: Inguinal hernia, cryptorchidism, genital dysplasia cleft palate and urachal fistula have already been described in patients with SCN4 3 . To this list we can now add renal agenesis, hydronephrosis (patient 2) and umbilical hernia (patient 4). Skeletal anomalies such as pectus carinatum, clinodactyly, broad thumbs and kyphosis could also be a part of this syndrome.

Additional dysmorphic features can provide useful clues towards a specific diagnosis.

Proper delineation of this aspect of the SCN4 phenotype will require a larger series of patients from different ethnic backgrounds. Considering the features observed in patient 4, it may be that mid-face hypoplasia, sparse scalp hair and loose skin represent features of SCN4. It is not clear if loose skin is an independent feature or secondary to failure to thrive.

In adolescents and adults, the fullness of cheeks is reminiscent of GSD1, and a truncal fat distribution might provide added clues to the diagnosis.

Conclusion

SCN4 is a recently described autosomal recessive condition whose phenotypic extent and natural history is still incompletely defined. It is important to recognize this syndrome so that appropriate treatment and monitoring can be instigated. Further studies into larger cohorts of patients are needed to determine the extent of possible complications, clarify associated dysmorphic features and determine if developmental delay and learning difficulties are consistent features of the syndrome. It is clear that significant inter-and intra-familial variability can occur. Delineation of the phenotype will also provide further insights into the mechanisms of this disease.

None

The highest and lowest available values of cell counts (prior to granulocyte colony stimulating factor administration, if treated) are given for each patient in multiples of 1000 cells per mm 3 . Counts which were detected to be outside the reference range on a majority of occasions have been underlined. Hb is given in g/dl with a reference range of 13.5-18.0 for adult males and for 11.5-16.0 for adult females. Reference range for HDL is 40 to 120 mg/ml; for uric acid is 2.6 to 6.0 mg/ml, for pancreatic amylase is 28 to100 U/L and for TSH is 0.4 to 4.0 IU/ml.

Cardiac anomalies :

 anomalies Patient 3 and 4 were diagnosed with PDA and secundum ASD. Patient 4 also had PS. Combining our data with that presented by Boztug et al and Jun et al, 13/19 patients have at least one congenital cardiac malformation (see table 2 for details). Secundum ASD and valvular defects are the most frequent cardiac anomalies in SCN4. Pulmonary vasculature anomalies: One patient in the series presented by Boztug et al had pulmonary venous malformation. The congenital cardiac malformations (PS, ASD and PDA) observed in patient 4 do not normally result in pulmonary hypertension in early life.

Figure 1 :

 1 Figure 1: Pedigrees of one large and one small nuclear family are represented. Both the

Figure

  Figure 2a and 2b: Front and profile of patient 2 showing full cheeks (similar to what is seen

Figure 3a :

 3a Figure 3a: Homozygous mutation c.758G>A in exon 6 of G6PC3 of Patient 1.

Figure 3b :

 3b Figure 3b: Corresponding normal sequence from a control subject.

Figure 1 IV

 1 Figure 1

  Key : B -Basophils, E -Eosinophils, Hb -Haemoglobin, HDL -High density lipoprotein cholesterol, L -Lymphocytes, M -Monocytes, MCH -mean corpuscular haemoglobin, MCV -Mean corpuscular volume, N -Neutrophils, P -Platelets, Pt -Patient, RC -Red cells, RR -Reference range, NA -Not available, TSH -Thyroid stimulating hormone and WC -White cells.

Table 1 :

 1 Summary of haematological and biochemical investigations of the four patients.

					Complete peripheral blood count				Significant results of other
		WC N	L	M	E	B	Hb	RC	MCV MCH P	Bone marrow aspiration	biochemical investigations
	RR	4.5	2.0	1.0	0.16	0	0	See	4.0	80	27	150	results	See foot-note
		to	to	to	to	to	to	foot-	to	to	to	to
		12.0	7.5	5.0	1.0	0.8	0.2	note	5.0	90 fl	31 pg	350
	Pt 1 2.0	0.7	2.2	0.2	0.01 0.01	11.4	4.5	77.3	25.1	38	Hypercellular marrow with	Reduced HDL -22 mg/ml.
		to	to	to	to		to	to	to	to	to	to	myeloid hyperplasia.	Elevated uric acid -7.2 mg/ml.
		4.0	1.3	2.3	0.4		0.02	13.0	4.9	81.8	26.5	140	Increased number of	Elevated pancreatic amylase -
													megakaryocytes and blast-	124 to148 U/L.
													like forms.	Raised TSH -4.73 IU/ml.
	Pt 2 1.7	0.2	1.1	0.17	0	0	9.9	3.87	77.5	24.0	233	NA	Raised TSH -4.99 IU/ml.
		to	to	to	to	to	to	to	to	to	to	to
		4.0	0.6	2.5	0.94	0.02	1.9	11.6	4.5	80.5	26.3	356
	Pt 3 2.1	0.2	0.9	0.3	0.02	0	10.8	4.5	75.0	23.2	260	Decreased erythropoesis,	None
		to	to	to	to	to	to	to	to	to	to	to	dysmyelopoetic changes
		3.2	1.5	1.7	0.8	0.12	0.01	12.5	4.9	81.9	25.2	428	with reduced granules in
													the cytoplasm and
													increased megakaryocytes.
	Pt 4 3.7	0.4	1.9	0.5	0.01	0.01	10.6	3.6	72.7	23.2	368	All stages of myelopoesis
		to	to	to	to	to	to	to	to	to	to	to	seen without any myeloid
		16.3	7.7	9.5	3.3	0.3	0.59	12.6	4.8	86.1	29.1	819	maturation arrest (See
													Supplemental Figure1)

Acknowledgements

The study was funded by the NIHR Manchester Biomedical Research Centre.

Conflict of interest

The authors declare no conflict of interest.

Supplementary information is available at The European Journal of Human Genetics

website.