Translation of a Research-Based Genetic Test on a Rare Syndrome into Clinical Service Testing,with Sotos Syndrome As an Example

Abstract

Background and Aims: It is often the case that the genetic background of a rare disease has been solved, but the testing of a clinical patient can be performed only through research projects. Translating a research-based test into diagnostic service may also appear laborious and costly. Based on our molecular research of the genetics of Sotos syndrome, we developed a clinical laboratory test that is both effective and relatively inexpensive. Methods and Results: Pilot testing was performed with samples of clinically diagnosed Sotos cases (n=13), and testing was continued with samples of patients who were suspected of having Sotos syndrome (n=161). The testing methods used were direct sequencing and multiplex ligation-dependent probe amplification. Sotos syndrome was a suitable example for test translation, because its genetic background was well established, and the demand for the test was expected to be fairly high. In the pilot phase, a mutation was detected in 12 out of 13 patients (92%), and in the second group, 49 out of 161 (30%) patients had a mutation in the NSD1 gene. Conclusions: In Sotos syndrome, detecting the mutation is valuable for the patient/family, while the value of a negative result is less clear and other differential diagnostic diagnoses should be considered. For successful translation of the research-based test into routine diagnostics, intense collaboration between clinicians, researchers, and diagnostic laboratory personnel is essential.

Introduction

New molecular genetic methods provide a vast spectrum of tools for detecting various mutations. Every diagnostic laboratory should evaluate which tests can be adopted from the research setting for diagnostics, and which are the factors that dictate the applicability of a given test in a diagnostic laboratory. In most laboratories, instrumentation is not the limiting factor, but the challenge is to determine the most cost-effective test, taking into consideration the utility of the test for patients and their families. In the era of genomics, research-based tests are being continuously developed, and the growing knowledge of new causative mutations creates a need for developing new diagnostic tests. However, different factors influence the translation of research-based testing into diagnostic testing: diagnostic laboratories have limited funds for test development, and they naturally prioritize tests for which a (relatively) high demand can be expected. On the other hand, researchers may not have close contact with diagnostic laboratories that are willing to proceed with test translation (Das et al., 2008; Faucett et al., 2008; Grody and Richards, 2008). Routine molecular diagnostics is particularly challenging in cases that involve a large gene or genes with various types of mutations, as is the case in many syndromes. Causative mutations can be scattered throughout the gene and can be of different types, from large-scale deletions to point mutations. There may also be genetic heterogeneity, often relating to only a small minority of cases, resulting in the screening of other possible genes also being cost ineffective. The syndrome itself may be relatively rare; nevertheless, it can often be suspected based on certain clinical findings.

Overgrowth and/or developmental delay are rather common clinical findings in pediatrics, and one among the syndromes that present with these features is the Sotos syndrome. The cardinal clinical features of the Sotos syndrome include macrocephaly, typical craniofacial features, developmental delay, and pre- and postnatal overgrowth (Cole and Hughes, 1994; Tatton-Brown et al., 2005). Different types of mutations of the large NSD1 gene are causative of the syndrome (Kurotaki et al., 2001, 2002). Several hundred intragenic point mutations as well as exonic deletions and microdeletions covering the large NSD1 gene have been described in the patients, and no mutational hotspot has been detected (Faravelli, 2005; Leventopoulos et al., 2009). Most cases are sporadic, but in a few families, autosomal dominant inheritance has been reported (Höglund et al., 2003; Tatton-Brown et al., 2005). A mutation can be found in ∼70%-90% of the patients with the clinical diagnosis, whereas a small proportion of the diagnosed patients are NSD1 mutation negative (Douglas et al., 2003; Rio et al., 2003; Türkmen et al., 2003; de Boer et al., 2005; Cecconi et al., 2005; Douglas et al., 2005; Tatton-Brown et al., 2005; Saugier-Veber et al., 2007). In non-Japanese patients, the most frequent mutation type is the intragenic point mutation, while Japanese patients more commonly carry a microdeletion (Kurotaki et al., 2002). It has been suggested that patients with microdeletions have more severe cognitive impairment and less pronounced overgrowth (Tatton-Brown et al., 2005).

In general, genetic tests, similar to all medical investigations, should be performed only if they are, at least to some extent, useful for the patient/family or health care. In the case of rare monogenic disorders, genetic testing is generally considered useful, as the result sets the diagnosis and facilitates the genetic counseling in the family. In literature, the test translation process has been discussed in the form of different guidelines, but rarely at a practical level. Guidelines and practices are truly valuable, because the growing knowledge of new syndromes and causative mutations creates a pressure for the diagnostic laboratories to offer testing outside research programs. We have used the Sotos syndrome as an example of the test translation process in our laboratories, and present here our experiences of the test translation at a practical level. We discuss the applicability of the molecular testing of the Sotos syndrome and present the outcome of testing to facilitate result interpretation in other diagnostic laboratories.

Patients and Samples

The pilot testing of the Sotos syndrome was performed with samples of 13 patients who were clinically diagnosed with Sotos syndrome by experienced clinicians (authors M.P. and M.T.P). After transferring the test to the diagnostic laboratory, samples from 161 patients have been referred for testing from several pediatric and genetic hospital units in Finland and Norway (data collected until Nov 2011). The criteria for testing were defined by the referring physician, and the main reasons were developmental delay and overgrowth, or a clinical diagnosis of Sotos syndrome. Informed consents and clinical data were obtained from the patients in the first cohort. In the second group, the patients were tested as a part of their clinical work-up, and the referring physician was, therefore, responsible for the consent according to local proceedings.

Molecular Methods

Multiplex ligation-dependent probe amplification

To detect exonic deletions and microdeletions encompassing the NSD1 gene, a multiplex ligation-dependent probe amplification (MLPA) analysis was performed with the P026B SALSA kit (MRC-Holland, Amsterdam, The Netherlands), which contains a probe for each NSD1 exon and two probes for the adjacent FGFR4 gene. The reactions were performed in a 0.25-fold volume of the reagents and 3-h hybridization modified from the manufacturer's protocol with 100 ng of genomic DNA. Samples from healthy individuals were used as negative controls, and samples with an NSD1 microdeletion were used as positive controls. The MLPA polymerase chain reaction (PCR) products were separated with an ABI Prism 310 capillary sequencer (Applied Biosystems, Foster City, CA), and the chromatograms were analyzed with the GeneMarker software (SoftGenetics, State College, PA). If single-exon deletions were detected, these exons were sequenced to detect possible point mutations in the probe binding site, causing reduction of the probe signals. All detected mutations were confirmed by re-performing the analysis with a stock sample.

PCR and sequencing

Intragenic point mutations in the NSD1 gene were analyzed with direct sequencing. All coding exons (2-23) and exon-intron boundaries were amplified in PCR with annealing temperatures of 55.5°C to 58.5°C (primers according to Douglas et al., 2003 with minor modifications, Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/gtmb) in a 25 μL reaction volume and using 100 ng of DNA. For sequencing, the reactions were purified with the ExoSAP enzyme mix (Fermentas, Burlington, Ontario, Canada). Purified PCR products were sequenced in both directions with the BigDye Terminator v.3.1 cycle sequencing kit (Applied Biosystems) and ABI Prism 310 and 3100 capillary sequencers (Applied Biosystems) according to the manufacturer's instructions. To confirm the mutations, the analysis was re-performed with a stock sample. For all mutations, nucleotides were numbered according to the GenBank cDNA reference sequence NM_022455.4, in which the “A” of the start codon is nucleotide 1. Mutation nomenclature was checked with mutalyzer sequence variant nomenclature checker v.1.0.1 (www.humgen.nl/mutalyzer/1.0.1/) according to the Human Genome Variation Society (HGVS) guidelines. Pathogenity prediction of missense mutations was performed using the PolyPhen (genetics.bwh.harvard.edu/pph) and Pmut (mmb.pcb.ub.es/PMut/) software.

Outcome of Genetic Analysis

Of the 13 patients diagnosed with classical Sotos syndrome in the pilot testing phase, 12 patients were discovered as having a mutation in the NSD1 gene (92%) and in one patient, no mutation was detected (8%). Of the second cohort of 161 patients referred for routine diagnostics and suspected of/diagnosed as having Sotos syndrome, mutations were detected in 49 patients (30%). In all, 58 pathogenic mutations and two possibly pathogenic variants were detected in 174 patients with a mutation rate of 33% (Table 1). Six deletions were detected using MLPA: four microdeletions covering the entire gene and two exonic deletions (exons 14 and 3-23). Fifty-five mutations were intragenic point mutations, and, of these, 34 mutations were either nonsense or frameshift mutations, causing a premature stop codon. Two mutations affected splice sites. In patient SOT80, the mutation occurred in the canonical splice site of exon 10, probably resulting in the skipping of exon 10. In patient SOT39a, a G→A transition 14 basepairs upstream of exon 21 created a cryptic splice site and caused an insertion of 13 basepairs into the mRNA. As a result, a premature stop codon was inserted 26 codons downstream from the mutation site. Nineteen missense changes were detected, and of them, 17 were located in functional domains and/or changed a conserved amino acid, and were considered pathogenic. Two of the variants detected, p.Cys1661Arg and p.Tyr1870Cys, were located outside the functional domains, but the computational pathogenity analysis predicted both of them to be pathogenic. In these cases, one or both parental samples were unavailable for testing, and the pathogenity remained elusive. Of all the mutations, 30 were novel and 23 were recurrent mutations that had been described in previous studies (Table 1). Recurrent mutations were also identified in this study: p.Arg604* was detected in three unrelated patients, and p.Ser1128Phefs*2 was found in two unrelated patients. One familial mutation (p.Ser299Tyrfs*21) was detected (Höglund et al., 2003).

Table 1.

The Mutations Detected in the NSD1 Gene in this Study

Mutation	Exon	Nucleotide change	Protein change	Domain	Parental status	Reference
Missense
SOT11	13	c.4786T>C	p.Cys1596Arg	PHD-2	NA
SOT141	13	c.4927T>G	p.Cys1643Gly	PHD-3	NA
SOT49	15	c.5173T>G	p.Cys1725Gly	PHD-3	NA
SOT174	15	c.5283G>C	p.Trp1761Cys	PWWP	NA
SOT48	18	c.5711C>G	p.Pro1904Arg	AWS	NA
SOT41a	18	c.5725T>C	p.Ser1909Pro	AWS	No mutation
SOT126	18	c.5842C>G	p.Arg1948Gly	SET	NA
SOT2a	18	c.5854C>T	p.Arg1952Trp	SET	No mutation	Tatton-Brown et al., 2005; Saugier-Veber et al., 2007
SOT70	19	c.5918G>T	p.Gly1973Val	SET	NA
SOT47	19	c.5927T>C	p.Ile1976Thr	SET	NA
SOT51	19	c.5951G>A	p.Arg1984Gln	SET	NA	Tatton-Brown et al., 2005; Waggoner et al., 2005; Saugier-Veber et al., 2007; Rio et al., 2003
SOT79	20	c.6050A>G	p.Arg2017Gln	SET	NA	Douglas et al., 2003; Tatton-Brown et al., 2005; Saugier-Veberet al., 2007; Rio et al., 2003
SOT90	20	c.6062A>G	p.His2021Arg	SET	NA	Saugier-Veber et al., 2007; Leventopoulos et al., 2009
SOT87	21	c.6221C>A	p.Ala2074Asp	post-SET	NA
SOT137	22	c.6356A>G	p.Asp2119Gly	PHD-4	NA	Waggoner et al., 2005
SOT162	22	c.6418A>G	p.Lys2140Glu	PHD-4	NA
SOT129a	23	c.6646G>A	p.Gly2216Arg	Pro-rich	No mutation
Nonsense
SOT5	5	c.2362C>T	p.Arg788^*		NA	Tatton-Brown et al., 2005; Saugier-Veber et al., 2007
SOT32	5	c.1318C>T	p.Arg440^*		NA	Rio et al., 2003
SOT78	5	c.1636C>T	p.Gln546^*		NA
SOT82	5	c.1810C>T	p.Arg604^*		NA	Douglas et al., 2003; Tatton-Brown et al., 2005; Waggoner et al., 2005
SOT83	5	c.1810C>T	p.Arg604^*		NA	Douglas et al., 2003; Tatton-Brown et al., 2005; Waggoner et al., 2005
SOT71	5	c.1810C>T	p.Arg604^*		NA	Douglas et al., 2003; Tatton-Brown et al., 2005; Waggoner et al., 2005
SOT29a	5	c.3067C>T	p.Arg1023^*		No mutation	Waggoner et al., 2005
SOT8	7	c.3958C>T	p.Arg1320^*		NA	Tatton-Brown et al., 2005; Saugier-Veber et al., 2007; Nagai et al., 2003
SOT3	10	c.4411C>T	p.Arg1471^*		NA	Douglas et al., 2003
SOT1c	16	c.5332C>T	p.Arg1778^*		No mutation	Douglas et al., 2003; Tatton-Brown et al., 2005; Saugier-Veber et al., 2007
SOT104	16	c.5431C>T	p.Arg1811^*		NA	Tatton-Brown et al., 2005; Waggoner et al., 2005; Saugier-Veber et al., 2007
SOT17c	17	c.5543T>G	p.Leu1848^*		No mutation
SOT34	22	c.6328C>T	p.Gln2110^*		NA
SOT123	22	c.6349C>T	p.Arg2117^*		NA	Tong et al., 2005
SOT16a	22	c.6349C>T	p.Arg2117^*		No mutation in the father	Tong et al., 2005
Frameshift
SOT26	2	c.896delC	p.Ser299Tyrfs^*21		Familial	Höglund et al., 2003
SOT6a	5	c.1658_1665dupGGATAGCA	p.Asn556Glyfs^*2		No mutation
SOT10	5	c.1864insT	p.Cys622Leufs^*7		NA
SOT85	5	c.2023_2024delAT	p.Met675Valfs^*7		NA
SOT13	5	c.2081_2091delCTAGGGTAAAA	p.Thr694Serfs^*8		NA
SOT116	5	c.2497delG	p.Val833Trpfs^*4		NA
SOT152	5	c.2658delA	p.Lys886Asnfs^*26		NA
SOT4a	5	c.2954_2955delCT	p.Ser985Cysfs^*25		No mutation	Waggoner et al., 2005; Saugier-Veber et al., 2007
SOT163	5	c.2956dupG	p.Ala986Glyfs'25		NA
SOT75	5	c.3004_3005delAA	p.Lys1002Gfs^*8		NA
SOT7	5	c.3280insA	p.Met1094Asnfs^*7		No mutation
SOT23a	5	c.3383_3384delCT	p.Ser1128Phefs^*2		No mutation in the mother	Douglas et al., 2003
SOT77	5	c.3383_3384delCT	p.Ser1128Phefs^*2		NA	Douglas et al., 2003
SOT106	5	c.3549insT	p.Glu1184 ^*		NA	Douglas et al., 2003; Waggoner et al., 2005
SOT103	6	c.3891insT	p.Gln1298Serfs^*16		NA
SOT131	8	c.4217_4220delGAAA	p.Arg1406Asnfs^*12		NA
SOT14a	8	c.4274_4277delAGAA	p.Lys1425Argfs^*20		No mutation
SOT72	12	c.4747_4748delTG	p.Cys1583Glnfs^*2		NA
SOT73	13	c.4827delC	p.Leu1610Cysfs^*32		NA
Splice site
SOT80	10	c.4497+1 G→A	ex 10 del		NA
SOT39	20	c.6010-14G>A	ex 20 del		No mutation
Exonic deletions
SOT50	14	c.4967-?_5146+?del	ex 14 del		NA	Piccione et al., 2011
SOT101	3-23	c.928-?_8091+?del	ex 3-23 del		No mutation
Microdeletions
SOT12	1-23	c.(?_-30)_(^*220)			NA
SOT28	1-23	c.(?_-30)_(^*220)			NA
SOT81	1-23	c.(?_-30)_(^*220)			NA
SOT161	1-23	c.(?_-30)_(^*220)			NA
Unclassified variants
SOT132	14	c.4981T>C	p.Cys1661Arg		NA
SOT76	17	c.5609A→G	p.Tyr1870Cys		No mutation in the father

NA, not analyzed.

Discussion

We have addressed the challenges involved in test translation in our laboratory by observing a close collaboration between the academic research and diagnostic laboratories, in addition to that close collaboration with the clinical genetics unit. The researchers receive requests/suggestions concerning new tests directly from clinicians, and the process usually begins with discussions between the clinicians and researchers with the aim of defining the characteristics of the test/syndrome in focus and the need for testing. For example, the Sotos syndrome was chosen as an example for the test translation, because the demand for the test was expected to be fairly high, due to the fact that the nonspecific symptoms of the syndrome are common in pediatrics, the specific facial features may be difficult for pediatricians to recognize, and the mutational background is well established. Once the pilot testing phase in the research laboratory begins, the diagnostic laboratory is involved from the very beginning in evaluating which methods are easily adapted for use in the diagnostic laboratory. The diagnostics laboratory also handles the research samples identically to the diagnostic samples, from sample registration and DNA extraction to the return of the result. Even in the pilot testing phase, our choice has been to return the research results to the patients through the clinicians involved, irrespective of the result itself. In the intermediate phase, the researchers and diagnostic laboratory personnel analyze the result generated in the research laboratory. After the diagnostic laboratory takes over the testing, the close collaboration of all parties still continues in order to ensure up-to-date knowledge at all levels of the testing procedure. For example, the researchers help the diagnostic laboratory in the interpretation of complicated findings.

Optimally, there should be a robust and relatively cheap method for mutation detection in all the diseases/situations where genetic testing is considered clinically useful. Sanger sequencing has been considered a practical choice for mutation detection in many rare diseases because of the high number of unique mutations in patients. As in rare diseases, the number of positive samples is low in the pilot phase, determination of the analytical validity and sensitivity of the method used may be difficult, and the use of a previously validated method may be advisable. In the pilot testing phase, to compare performance and costs, we sought intragenic mutations in parallel with direct sequencing and the prescreening method denaturing high-performance liquid chromatography (DHPLC). Based on the results, we chose direct sequencing as the method to be used in Sotos syndrome diagnostics. The reasons for this were that DHPLC was not routinely used in the diagnostics laboratory, and the setting up and maintenance of the DHPLC machinery was not deemed expedient and suitable for the methodological strategy of the diagnostics laboratory. In addition, the sensitivity and specificity of sequencing were higher than in DHPLC. Even though costs of direct sequencing of the large NSD1 gene exceed the costs of prescreening, by choosing the sequencing as the method to be used in diagnostics, a compromise was made between the costs, the workload, and the feasibility of the laboratory setting up of a new test.

Since a small percentage of the NSD1 mutations are exonic deletions or microdeletions, another method was needed to detect these mutations. For this purpose, the alternative methods considered were MLPA and array comparative genomic hybridization (CGH). In terms of difference, an MLPA test covers only the NSD1 gene, while array CGH usually covers the whole genome. Arrays are often used in the diagnostics of rare syndromes as a primary test, especially when no particular syndrome is suspected, but we chose MLPA because of the simple set up and robustness of the method, allowing the adaptation of the method also to other diseases tested in the diagnostic laboratory. To confirm the Sotos syndrome diagnosis, the detection of an NSD1 deletion is sufficient, because no other genes are known to be causative of this syndrome. From this point of view, since our test aims particularly at confirming or excluding the Sotos syndrome, the size of the deletion is irrelevant. However, if the symptoms of the patient indicate a large deletion, array CGH can then be used to define the size of the deletion more precisely. This may be of consequence, as large genomic rearrangement will alter the prognosis to the extent that the definition of the individual's genomic status is recommendable. In addition, the test specific for the Sotos syndrome does not reveal other possible genomic imbalances leading to a different diagnosis.

Before a given test is used in diagnostics, it should be validated for its use. For example, a test might perform superbly in the laboratory, but its use in diagnostics is limited. Alternatively, a test that performs poorly in the laboratory might have an enormous impact on patient health (Anna Dierking, personal communication). To examine the usefulness of the test in diagnostics, parameters such as analytical validity, clinical validity, and clinical utility can be utilized. The analytical validity of the Sotos syndrome testing is close to 100%, if both sequencing and MLPA are used. Without MLPA or any other deletion detection method, a minority of mutations remains undetected, degrading the analytical validity of the test. The clinical sensitivity of the test, according to the clinical criteria of Cole and Hughes (1994), is ∼90%. In ∼10% of the clinically diagnosed patients, the pathogenic mutation probably locates outside the NSD1 coding area, and, therefore, will remain undetected (Douglas et al., 2003; Tatton-Brown et al., 2005). The clinical specificity of the test is ∼95%, because a small number of patients with atypical features carry a mutation in the NSD1 gene, and their mutation will not be found if strict clinical criteria are used (Tatton-Brown et al., 2005). Most importantly, the criteria for the clinical utility of Sotos syndrome testing are clearly fulfilled (Javaher et al., 2008). The NSD1 defect is confirmatory, and its absence is highly suggestive of another diagnosis. Confirmation of a diagnosis is extremely important to the psychosocial health of a family, and it also signifies the end of the stressful search for diagnosis with different, often invasive, methods. The natural history of the Sotos syndrome is fairly well known and recommendations for follow-up exist, which are important issues in the counseling of the family (Cole, 2005).

By November 2011, we had tested 174 patients with diagnosed or suspected Sotos syndrome, and detected a mutation in 58 patients (33%). Most of the mutations were nonsense and frameshift mutations, leading to a formation of premature stop codons, and a minority of the mutations were missense, splice site, intragenic, or whole NSD1 deletions, correlating with previously published reports. The difference in the mutation rates between the two cohorts in our study—92% in the cohort of clinically diagnosed patients and 30% in the cohort of routine diagnostic patients—reflects the challenges in diagnosing the Sotos syndrome and use of the test in differential diagnostics. Many clinicians meet patients with the Sotos syndrome only occasionally, and in these situations, diagnostic testing is particularly necessary. For an experienced clinician, the correlation between clinical diagnosis and the presence of mutations in the NSD1 gene is close to 100%, but the experienced clinicians may not have the need to have their clinical diagnosis confirmed with mutation testing, causing a bias in the mutation detection percentage in the diagnostic cohort.

In the future, the introduction of the next-generation sequencing (NGS) technology will evoke an entirely new approach to the diagnostics of rare diseases. NGS reports presenting new mutations behind new and known clinical syndromes that are accumulating, and laboratories are beginning to set up NGS for diagnostic use. Conventional diagnostics has been extremely difficult; for example, in the case of unspecific mental retardation, movement disorders, and blindness, due to the large number of candidate genes and rare private variants. In these situations, NGS will have an enormous advantage compared with contemporary methods. However, disorders with single gene mutations or recurrent mutations are outside the focus of NGS for the present. In the future, the diagnostic decision-making may be begun only after the clinician has the NGS data in use, but until NGS becomes a routine method, the current and newly developed diagnostic tests are still needed.

As a conclusion, translation of research-based tests into diagnostic testing can be challenging, but it can be performed successfully with a close collaboration of research and diagnostic laboratories and clinical genetics units. The choice of the syndrome to be tested and the methods to be used are dependent on the mutational background of the syndrome in focus and the genotype-phenotype correlation. In addition, the clinical utility and cost efficiency in the routine diagnostics laboratory have to be considered. We have used the Sotos syndrome as an example for test translation and have now tested 174 samples with a 33% mutation detection percentage. Testing is particularly helpful in situations in which clinicians have little experience in Sotos syndrome to confirm or exclude the clinical diagnosis.

Footnotes

Acknowledgments

The authors would like to thank all the patients and families who participated in their study, as well as all referring clinicians, Damon and Maaria Tringham, for language revision, Petra Hämäläinen and Minna Toivonen for providing the mutation data and helpful discussions, and Professor Egbert Bakker and the personnel in the Leiden University Medical Center Sylvius Laboratory for an MLPA training session and providing the MLPA protocol. This study was funded by the Special Federal Grant of Turku University Central Hospital and the University of Turku Foundation and EuroGentest2, an EU-FP7 supported coordination action (contract number HEALTH-F4-2070-261469). The Department of Medical Genetics of The Family Federation of Finland is funded by Finland's Slot Machine Association (RAY).

Author Disclosure Statement

The authors state that no competing financial interests exist.

References

Cecconi

, Forzano

, Milani

et al. 2005. Mutation analysis of the NSD1 gene in a group of 59 patients with congenital overgrowth. Am J Med Genet A, 134:247-253.

Cole TRP (2005) Sotos Syndrome. Cassidy

, Allanson

. Management of Genetic Syndromes, 2nd. Hoboken: New Jersey, 527-538.

Cole

, Hughes

. 1994. Sotos syndrome: a study of the diagnostic criteria and natural history. J Med Genet, 31:20-32.

Das

, Bale

, Ledbetter

. 2008. Molecular genetic testing for ultra-rare diseases: models for translation from the research laboratory to the CLIA-certified diagnostic laboratory. Genet Med, 10:332-336.

de Boer

, Kant

, Karperien

et al. 2004. Genotype-phenotype correlation in patients suspected of having Sotos syndrome. Horm Res, 62:197-207.

Douglas

, Hanks

, Temple

et al. 2003. NSD1 mutations are the major cause of Sotos syndrome and occur in some cases of Weaver syndrome but are rare in other overgrowth phenotypes. Am J Hum Genet, 72:132-143.

Douglas

, Tatton-Brown

, Coleman

et al. 2005. Partial NSD1 deletions cause 5% of Sotos syndrome and are readily identifiable by multiplex ligation dependent probe amplification. J Med Genet, 42:e56.

Faravelli

. 2005. NSD1 mutations in Sotos syndrome. Am J Med Genet C Semin Med Genet, 137:24-31.

Faucett

, Hart

, Pagon

et al. 2008. A model program to increase translation of rare disease genetic tests: collaboration, education, and test translation program. Genet Med, 10:343-348.

10.

Grody

, Richards

. 2008. New quality assurance standards for rare disease testing. Genet Med, 10:320-324.

11.

Höglund

, Kurotaki

, Kytölä

et al. 2003. Familial Sotos syndrome is caused by a novel 1 bp deletion of the NSD1 gene. J Med Genet, 40:51-54.

12.

Javaher

, Kääriäinen

, Kristoffersson

et al. 2008. EuroGentest: DNA-based testing for heritable disorders. Community Genet, 11:71-120.

13.

Kurotaki

, Harada

, Yoshiura

et al. 2001. Molecular characterization of NSD1, a human homologue of the mouse Nsd1 gene. Gene, 279:197-204.

14.

Kurotaki

, Imaizumi

, Harada

et al. 2002. Haploinsufficiency of NSD1 causes Sotos syndrome. Nat Genet, 30:365-366.

15.

Leventopoulos

, Kitsiou-Tzeli

, Psoni

et al. 2009. Three novel mutations in Greek Sotos patients with rare clinical manifestations. Horm Res, 71:45-51.

16.

Nagai

, Matsumoto

, Kurotaki

et al. 2003. Sotos syndrome and haploinsufficiency of NSD1: clinical features of intragenic mutations and submicroscopic deletions. J Med Genet, 40:285-289.

17.

Piccione

, Consiglio

, Di Fiore

et al. 2011. Deletion of NSD1 exon 14 in Sotos syndrome: first description. J Genet, 90:119-123.

18.

Rio

, Clech

, Amiel

et al. 2003. Spectrum of NSD1 mutations in Sotos and Weaver syndromes. J Med Genet, 40:436-440.

19.

Saugier-Veber

, Bonnet

, Afenjar

et al. 2007. Heterogeneity of NSD1 alterations in 116 patients with Sotos syndrome. Hum Mutat, 28:1098-1107.

20.

Tatton-Brown

, Douglas

, Coleman

et al. 2005. Genotype-phenotype associations in Sotos syndrome: an analysis of 266 individuals with NSD1 aberrations. Am J Hum Genet, 77:193-204.

21.

Tong

, Hau

, Lo

et al. 2005. Spectrum of NSD1 gene mutations in southern Chinese patients with Sotos syndrome. Chin Med J (Engl), 118:1499-1506.

22.

Türkmen

, Gillessen-Kaesbach

, Meinecke

et al. 2003. Mutations in NSD1 are responsible for Sotos syndrome, but are not a frequent finding in other overgrowth phenotypes. Eur J Hum Genet, 11:858-865.

23.

Waggoner

, Raca

, Welch

et al. 2005. NSD1 analysis for Sotos syndrome: insights and perspectives from the clinical laboratory. Genet Med, 7:524-533.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.03 MB