Dear Editor

Mattocks et al [1] have used direct DNA sequencing and comparative sequence analysis to study NF1 patients and claim this study “achieved the highest recorded mutation detection rate using a single technique for this gene”. As a key point, the paper states that they studied 91 subjects fulfilling the NIH NF1 diagnostic criteria and achieved a mutation detection rate of 89% using automated comparative sequence analysis. They continue by saying “This detection rate is the highest for a single technique and is therefore appropriate for routine clinical practice”.

When developing genetic tests, especially for large and complex genes such as NF1, a large cohort of patients needs to be studied in a comprehensive way in order to fully understand the spectrum of mutations present in that gene. From our experience, it is of utmost importance to analyze the complete gene for the presence of all possible alterations that may result in a premature stop codon at the mRNA level [2]. A significant fraction of the mutations in the NF1 gene cause aberrant splicing and many of them are due to alterations outside the canonically conserved AG/GT acceptor and donor sequences and even reside deep into the large introns [2-4]. Also, a number of exonic mutations mimicking nonsense, missense and even silent mutations at the genomic level have been described that are splicing mutations and exert their effect by creating a novel splice donor or acceptor or affect the function of an exonic splicing enhancer (ESE) or exonic splicing silencer (ESS) [2, 5] . Although we have now studied over 600 patients fulfilling the NIH criteria using multiple complementary techniques, we are still challenged and surprised by the diversity of mutations leading to this disorder.

There have been, unfortunately, some examples in literature where an alteration is claimed to be a pathogenic mutation, and where later on this statement needs to be revoked as the alteration is proven to be an innocent polymorphism [6]. For thousands of hereditary disorders for which the genes have been cloned, patients await the availability of a reliable and sensitive diagnostic test and clinical molecular genetic labs worldwide rely on published reports to help distinguish a polymorphism or rare benign variant from a deleterious mutation. This distinction is of utmost importance and has major ethical implications with respect to the genetic counseling of patients seeking diagnostic, pre-/oligosymptomatic and prenatal testing.

There is a need for a reliable and sensitive genetic test for the NF1 gene, to help resolve diagnostic dilemmas in patients not fulfilling the NIH diagnostic criteria, especially young children but also atypical patients, to determine the affection status of family members of an affected person and to perform prenatal or pre-implantation diagnosis, if desired. NF1 is a progressive disorder and many features increase in frequency with age. CAL-spots are often the first signs of NF1 and may already be present at birth, increasing in number during the first years of life. Only about half of patients with sporadic NF1 fulfill the NIH diagnostic criteria by one year of age and still 5% will not fulfill these criteria by age 8 years [7]. Waiting for more symptoms to appear with time in order to ascertain the diagnosis on a clinical basis can be very stressful for families. Earlier diagnosis of NF1 allows to offer genetic counseling to parents and relatives earlier as well as to initiate interventions for learning or developmental problems sooner. Earlier diagnosis will become even more important once more therapeutic options become available. A direct genetic test may help to establish the diagnosis earlier, especially in sporadic patients, but only when the testing has a high sensitivity, i.e. finds the mutation in (almost) all patients that eventually will fulfill the NIH criteria (low false negative results) and, equally important, does not confuse a benign variant with a pathogenic mutation (no false positive results).

We have identified multiple sequence changes in the paper by Mattocks et al that are misclassified and hence need rectification to avoid potential misdiagnoses based on the latter information. Whereas the title of the paper states that automated comparative sequence analysis identifies mutations in 89% of NF1 patients, Table 2 describes these sequence alterations as “potentially significant” sequence alterations. There also is a Table 3 summarizing polymorphisms found in the study, which adds to the confusion and further suggests that alterations in Table 2 are pathogenic mutations, as also the title alludes. We think we need to make a clear distinction between a deleterious mutation, an unclassified variant, a rare benign variant and a polymorphism.

Table 2 contains four silent nucleotide changes: Q282Q, C680C, K1724K and R1808R. The authors predict that these changes lead to a truncated peptide, but no experimental evidence is given to proof this. Two of these sequence changes have been observed by us and others and are definite rare benign variants. c.5172G>A (K1724K) was first described by Peters et al [8] as a polymorphism with allele frequencies of 0.99 for c.5172G and 0.01 for c.5172A. Peters et al [8] report on a patient who carries this polymorphism in exon 28 as well as a frameshift mutation in exon 28. Also, Fahsold et al [9] describe a patient with the c.5172G>A sequence change as well as a pathogenic frameshift mutation in exon 37: c.6789del4. One of us (LM) has observed this sequence change in 2/570 NF1 patients in which a clearly pathogenic mutation, i.e. c.3216delC and c.1756_1759delACTA was found as well. Thus, this sequence variant represents an infrequent benign variant that should not be confused with a bona fide pathogenic mutation. Mattocks et al demonstrated that the silent nucleotide alteration K1724K was also found in the affected mother and, thus, segregated with the disease. However, this example clearly shows that this is not sufficient to render final evidence of whether a variation is pathogenic. An effect on splicing should be evidenced before a silent change should be classified pathogenic. A second silent sequence change Q282Q (c.846G>A) predicted by Mattocks et al to result in a truncated protein has been described by Luca et al [10] to be a polymorphism with a frequency of 2 %. One of us (LM) also found Q282Q in 2/190 control samples and hence this alteration also has to be considered as a rare benign variant. By protein truncation testing and direct cDNA sequencing as described previously [2], we did not observe production of a truncated peptide nor any effect on splicing due to Q282Q. In the light of these obvious misclassifications, the prediction of the truncating effect of the two other silent sequence changes (c.2040C>T C680C; c.5427G>A R1808R) needs to be considered with great caution, especially as these changes fulfill none of the classic criteria for pathogenic mutations: both silent changes have not been reported previously, they have not been demonstrated to occur de novo in sporadic patients neither were they shown to segregate with the disease in a given family and most importantly they were not proven to affect splicing. Hence, these changes can not be considered pathogenic unless data are provided rendering evidence that these changes have an effect on the correct splicing of the NF1 gene. Through the study of over 600 unrelated NF1 patients fulfilling NIH criteria, we identified 29 patients carrying a pathogenic truncating mutation as well as a silent mutation, the latter without observed effect on splicing (Messiaen et al, unpublished results).

Apart from the silent sequence changes, also the classification of NF1 missense mutations is particularly challenging. Table 2 contains at least one missense alteration (D176E) that was reported previously to be a polymorphism [9]. One of us (LM) also identified D176E in 1 NF1 patient carrying another clearly pathogenic alteration and in 1/190 normal control samples, confirming D176E is indeed a rare benign variant. We do not understand why the authors list a patient carrying this sequence change in Table 2, since in their Table 3 they state that this alteration is a polymorphism also found in unaffected individuals. Similarly, they list a patient carrying the missense alteration c.2617C>T (R873C) in Table 2, while at the same time state in Table 3 that this also is a benign variant they found in a patient who carried a clearly pathogenic mutation c.1- 14_7del21bp. Nevertheless, the authors mention both alterations in Table 2 and these data as well as the formerly mentioned misclassified alterations are taken into account to come to the conclusion that the technique has a 89% detection rate. Y489C (c.1466A>G) is one of the most frequent recurrent mutations in NF1 patients and was the first well-understood splice mutation that could be misclassified as a missense mutation if only genomic DNA would be studied [11]. This mutation results in the creation of a perfect novel splice donor that is used by the splicing machinery instead of the wild- type exon 10b donor, and leads to skipping of the last 62 nucleotides of exon 10b. Y489C has since been reported in many NF1 mutation papers [9, 12 -14]. Hence we do not understand why Mattocks et al describe this mutation, which they found in 3 NF1 patients, as a missense mutation that was not previously reported. G629R (c.1885G>A) can not be considered to be purely a missense mutation either: Ars et al reported a splice effect in 5 patients [3]. We observed this splice effect in 3 unrelated patients as well (Messiaen et al, unpublished results): the observed splicing error is readily understood by the creation of a novel splice acceptor site by this mutation, leading to skipping of the first 41 bp of exon 12b.

Recalculating the number of putative missense mutations after subtraction of the above mentioned misclassifications, Table 2 of the Mattocks et al. paper still contains a total of 12 different missense mutations or small deletions of 1 or 2 amino acids that affect 15 patients fulfilling the NIH diagnostic criteria, i.e H31R, L145P, E337V, C324R, L532P, S574R, L844P, R1276G, R1276Q, ÄE1438, ÄIY1658-9 and ÄNF2366-7. Hence, as many as 16.5% (15/91) of the patients fulfilling the NIH diagnostic criteria, harbor putative missense mutations and small deletions of one or more amino acids. This number appears quite high in comparison to other previous reports with high mutation detection rates (Messiaen et al. 2000, Ars 2000). Thus, it may very well be that a portion of the novel missense mutations found in the study of Mattocks at al are splicing mutations. The authors are aware of this possibility and have developed tools such as a functional splicing assay using a minigene system to test for the effect on splicing. We do not understand why they did not apply these tools in this study to achieve a conclusion on the effect of silent and missense mutations. Furthermore, some of the novel missense mutations -similarly as D176E, R873C, A2058D - may turn out to be non pathogenic rare sequence variants. Their finding of different missense mutations (R873C and A2058D) in 2 NF1 patients carrying another clearly pathogenic mutation further underscores this possibility.

In the absence of functional assays, rigorous criteria must be applied before a novel missense alteration in the NF1 gene can be classified as the disease causing mutation in order to avoid diagnostic errors. The following criteria are proposed and applied when clinical testing is offered: i/ absence of any other possible deleterious mutation after analysis of the whole coding region. Analysis must include screening for a total gene deletion, smaller deletions (one to multiple exons deletions), splice mutations including deep intronic mutations affecting splicing. This is not achieved when only genomic DNA is studied as described in the paper by Mattocks et al. If RNA-based mutation analysis reveals an effect on splicing, the “missense” mutation can be considered to be deleterious; ii/ absence of the sequence alteration in a large number of unrelated control samples. This is a necessary but insufficient criterion. Indeed, we still find novel benign variants on the wild-type NF1 gene, inherited from the unaffected parent, even after analyzing >600 patients; iii/ support from evolutionary conservation in Mus musculus, Rattus norvegicus, Takifugu rubripes and Drosophila melanogaster of the amino acid under consideration as well as support from algorithms such as the ones developed by Miller and Kumar [15]; iv/ finally and importantly, clinical and molecular genetic assessment of the family. In case the patient is a sporadic case, the missense mutation needs to be proven to be a de novo event and clinical evaluation of both parents needs to show absence of the disorder in them. In case the patient has a positive family history, the missense mutation needs to be proven to segregate with the disorder in the family by analysis of one affected relative.

Taken together, the two main findings summarized in the title of the paper by Mattocks et al do not withstand a critical review of the data provided. We believe it is not justified to draw conclusions on detection rates of the assay presented here unless the pathogenity of the novel silent and missense alterations contained in Table 2 has been evidenced by some means. After pointing out a number of obvious misclassifications in the list it is fair to state that the detection rate is certainly lower than calculated by the authors. Furthermore, due to these misclassifications and the lack of evidence that a fraction of the remaining missense alterations do not affect splicing it is not justified to use these data to confirm or refute a mutation cluster in the exons 11-17 pointing to the existence of a novel functional domain..

We disagree that this single technique as applied here is appropriate for clinical practice and strongly advocate comprehensive analysis of the complete coding region before a missense or silent alteration is considered to be a pathogenic alteration. In the absence of any other possible pathogenic alteration, missense mutations must be evaluated according to the criteria discussed above. Silent mutations can not be considered to be pathogenic unless proof is provided that they are altering function, e.g. correct splicing. We are hopeful and optimistic that mutation detection in the NF1 gene will become more easily accomplished, faster and cheaper once more reliable data become available and technologies further develop. Then, direct cycle sequencing or resequencing arrays will allow detecting in a fast and efficient manner a fair number of pathogenic lesions. However, genomic DNA sequencing assays alone will not allow deciding on the pathogenicity of missense or silent alterations, not yet described before. This reply is meant to stimulate vigilance in the community to avoid potential diagnostic errors.

Ludwine Messiaen (1) and Katharina Wimmer (2)

(1) Dept of Genetics, University of Alabama at Birmingham, US.

(2) Dept of Human Genetics at the Clinical Institute for Medical and Chemical Laboratory Diagnostics, Medical University Vienna, Austria.

References

(1) Mattocks C, Tarpey P, Bobrow M, Whittaker J. Comparative sequence analysis (CSA): A new sequence-based method for the identification and characterization of mutations in DNA. Hum Mutat 2000 Nov;16(5):437-43.

(2) Messiaen LM, Callens T, Mortier G, Beysen D, Vandenbroucke I, Van Roy N, Speleman F, Paepe AD. Exhaustive mutation analysis of the NF1 gene allows identification of 95% of mutations and reveals a high frequency of unusual splicing defects. Hum Mutat 2000;15(6):541-55.

(3) Ars E, Serra E, Garcia J, Kruyer H, Gaona A, Lazaro C, Estivill X. Mutations affecting mRNA splicing are the most common molecular defects in patients with neurofibromatosis type 1. Hum Mol Genet 2000 Jan 22;9(2):237-47.

(4) Perrin G, Morris MA, Antonarakis SE, Boltshauser E, Hutter P. Two novel mutations affecting mRNA splicing of the neurofibromatosis type 1 (NF1) gene. Human Mutation 1996;7(2):172-5.

(5) Zatkova A, Messiaen L, Vandenbroucke I, Wieser R, Fonatsch C, Krainer AR, Wimmer K. Disruption of exonic splicing enhancer elements is the principal cause of exon skipping associated with seven nonsense or missense alleles of NF1. Hum Mutat 2004 Nov 2;24(6):491-501.

(6) Lambert J, Naeyaert JM, De Paepe A, Van Coster R, Ferster A, Song M, Messiaen L. Arg-Cys Substitution at Codon 1246 of the Human Myosin Va Gene is not Associated with Griscelli Syndrome. J Invest Dermatol 2000 Apr 1;114(4):731-3.

(7) DeBella K, Szudek J, Friedman JM. Use of the national institutes of health criteria for diagnosis of neurofibromatosis 1 in children. Pediatrics 2000 Mar;105(3 Pt 1):608-14.

(8) Peters H, Luder A, Harder A, Schuelke M, Tinschert S. Mutation screening of neurofibromatosis type 1 (NF1) exons 28 and 29 with single strand conformation polymorphism (SSCP): five novel mutations, one recurrent transition and two polymorphisms in a panel of 118 unrelated NF1 patients. Mutations in brief no. 229. Online. Hum Mutat 1999;13(3):258.

(9) Fahsold R, Hoffmeyer S, Mischung C, Gille C, Ehlers C, Kucukceylan N, Abdel-Nour M, Gewies A, Peters H, Kaufmann D, Buske A, Tinschert S, Nurnberg P. Minor lesion mutational spectrum of the entire NF1 gene does not explain its high mutability but points to a functional domain upstream of the GAP-related domain. Am J Hum Genet 2000 Mar;66(3):790-818.

(10) De Luca A, Buccino A, Gianni D, Mangino M, Giustini S, Richetta A, Divona L, Calvieri S, Mingarelli R, Dallapiccola B. NF1 gene analysis based on DHPLC. Hum Mutat 2003 Feb;21(2):171-2.

(11) Messiaen L, Callens T, Roux K, Mortier G, de Paepe A, Abramowicz M, Pericak-Vance M, Vance J, Wallace MR. Exon 10b of the NF1 gene represents a mutational hotspot and harbors a recurrent missense mutation Y489C associated with aberrant splicing. Genet Med. 1(6), 248- 253. 2004. Ref Type: Generic

(12) Ars E, Kruyer H, Morell M, Pros E, Serra E, Ravella A, Estivill X, Lazaro C. Recurrent mutations in the NF1 gene are common among neurofibromatosis type 1 patients. J Med Genet 2003 Jun;40(6):e82.

(13) Han SS, Cooper DN, Upadhyaya MN. Evaluation of denaturing high performance liquid chromatography (DHPLC) for the mutational analysis of the neurofibromatosis type 1 ( NF1) gene. Hum Genet 2001 Nov;109(5):487- 97.

(14) Osborn MJ, Upadhyaya M. Evaluation of the protein truncation test and mutation detection in the NF1 gene: mutational analysis of 15 known and 40 unknown mutations. Hum Genet 1999 Oct;105(4):327-32.

(15) Miller MP, Kumar S. Understanding human disease mutations through the use of interspecific genetic variation. Human Molecular Genetics 2001 Oct 1;10(21):2319-28.

Dear Editor              

We thank the authors for their comments on our Letter to the Editor describing a novel locus for autosomal dominant keratoconus on chromosome 3 [1]. We thoroughly agree with the author’s opinion that simple astigmatism should not be considered a diagnostic criteria for forme fruste KC. However, none of our 4 patients diagnosed with forme fruste KC had simple astigmatism. In these patients, the clinical diagnosis of KC was based on a 1,5 Dioptre (D) increment scale videokeratographic analysis. In particular, in patient III:9 (whose videokeratographic picture is shown in the middle panel of figure 2) astigmatism was irregular on the right eye and regular on the left. A superior-inferior difference was recorded in both eyes, while in the right the corneal apex was dislocated in the inferior temporal quadrant of the cornea. Thus, this patient fulfil the diagnostic criteria for forme fruste KC [2]. Figure 2 showed these findings, although some details could have been lost in the printed version. We confirmed these data in patient III:9 with a more sensitive quantitative evaluation of the same videokeratography using a colorcoded map with 0,45 D increments. This analysis showed that the highest keratometric values in dioptre were 42,70 D on the superior and 43,60 D on the inferior cornea of the right eye and 43,60 D in the superior and 45,0 D on the inferior cornea of the left eye. Taken together these data point out the role of videokeratography in the diagnosis of subtle corneal abnormalities such as forme fruste KC, in agreement with Levy and coworkers who recommend to achieve a detection rate of at least 0.5 D increment scale for forme fruste KC [3].                              

Finally, the Hudson-Stahli’s line, described in two KC patients in table 2 was not considered as a sign of the disease but an occasional finding on slit-lamp examination.

References

(1) Liskova P. Diagnostic criteria of forme fruste keratoconus [electronic response to Brancati F et al. A locus for autosomal dominant keratoconus maps to human chromosome 3p14–q13] 

(2). Brancati F, Valente EM, Sarkozy A, Feher J, Castori M, Del Duca P, Mingarelli R, Pizzuti A, Dallapiccola B. A locus for autosomal dominant keratoconus maps to human chromosome 3p14-q13. J Med Genet 2004;41:188- 192.

(3). Rabinowitz YS. Videokeratographic indices to aid in screening for keratoconus. J Refract Surg 1995;11:371-379. 4. Levy D, Hutchings H, Rouland JF, Guell J, Burillon C, Arne JL, Colin J, Laroche L, Montard M, Delbosc B, Aptel I, Ginisty H, Grandjean H, Malecaze F. Videokeratographic anomalies in familial keratoconus. Ophthalmology 2004;111:867-874.

The authors: Francesco Brancati1,2, Enza Maria Valente1, Anna Sarkozy1,2, Jànos Fehèr3, Marco Castori1,2, Pietro Del Duca4, Rita Mingarelli1, Antonio Pizzuti1,2 and Bruno Dallapiccola1,2

1 CSS Hospital, IRCCS, San Giovanni Rotondo and CSS-Mendel Institute, Rome;

2 Department of Experimental Medicine and Pathology, University “La Sapienza”, Rome; 

3 Department of Ophthalmology, University "La Sapienza", Rome;

4 U.O. Internal Medicine, POC, Montefiascone, Viterbo.

Dear Editor

We would like to comment on the letter describing linkage analysis in autosomal dominant keratoconus presented by Brancati et al. in the March issue of JMG [1]. The pedigree includes 4 patients with forme fruste keratoconus and Figure 2 shows the videokeratography of patient III:9 to support this diagnosis. However, from the data presented this patient appears to have unilateral simple astigmatism, which does not fulfil the diagnostic criteria defined in the reference provided [2]. Futhermore, there is little evidence of inferior corneal steepening and the keratometry values are not unusually high [2,3] Corneal thickness measurements, which may be reduced in keratoconus, were not presented.

The authors point out that large pedigrees with clinically evident keratoconus are rare and videokeratography has been used to identify asymptomatic family members who have early changes of keratoconus. These patients with forme fruste keratoconus allow pedigrees to be expanded [4,5]. Levy et al. (2004) identified specific parameters that may characterise forme fruste keratoconus [6]. However it has yet to be shown that these abnormalities inevitably progress to clinical keratoconus .We consider that the diagnostic criteria for forme fruste keratoconus should not be expanded to include patients with simple astigmatism until a link between the two conditions can be established. The exclusion of patient III:9 from the study would inevitably result in a reduction in the significance of the calculated LOD score.

In addition, we would also like to point out that the clinical sign of a Hudson Stahli line used to describe the clinical features of the disease in affected patients (Table 2) is usually accepted to be a sign of a normal cornea. A circular Fleischer ring is the clinical sign that accompanies the central corneal steepening of keratoconus

Although this letter raises the interesting possibility of a new locus for familial keratoconus the points that we have highlighted should be addressed to clarify the diagnosis of the early stages of the disease.

References

(1) Brancati F, Valente EM, Sarkozy A, et al. A locus for autosomal dominant keratoconus maps to human chromosome 3p14-q13. J Med Genet 2004;41(3):188-92.

(2) Rabinowitz YS. Videokeratographic indices to aid in screening for keratoconus. J Refract Surg 1995;11(5):371-9.

(3) Rabinowitz YS. The genetics of keratoconus. Ophthalmol Clin North Am 2003;16(4):607-20, vii.

(4) Rabinowitz YS. Keratoconus. Surv Ophthalmol 1998;42(4):297-319.

(5) Morrow GL, Stein RM, Racine JS, et al. Computerized videokeratography of keratoconus kindreds. Can J Ophthalmol 1997;32(4):233 -43.

(6) Levy D, Hutchings H, Rouland JF, et al. Videokeratographic anomalies in familial keratoconus. Ophthalmology 2004;111(5):867-74.

Dear Editor

We read with interest a recent report in your journal by Jakkula et al.[1] on two families of multiple epiphyseal dysplasia (MED) with the recurrent R718W mutation in COMP.

Key points of the report are:
1) In a family (family 1), two children presented with muscular weakness and all four family members with R718W showed moderate rise in plasma creatine kinase (CK), indicating possible association of myopathy;
2) In both families, the skeletal change is more severe in the knee joint than in the hip joint. This observation contradicts to the previous assumption of genotype-phenotype association, i.e. presence of dysplastic capital femoral epiphyses and severely irregular acetabuli is suggestive of COMP mutations, while dysplastic changes in patients with collagen IX mutation are mainly seen in the knees and the hips are relatively spared.[2,3]

We previously experienced a MED family with three affected individuals harboring the same R718W mutation.[4] Here we describe their clinical and radiographic features for comparison and further discussion.

The proband was a 64-year-old factory laborer who presented with bilateral coxalgia at aged 56 years that had started 2 years. His height was 152 cm (nearly Ð3 SD), and his weight 53.5 kg. He had no history of muscle weakness. Physical examination had no indication of myopathy. His CK was 67 (normal range: 50-170) U/L, and was kept within the normal range during the follow-up period. Radiographic examination showed severe osteoarthritic (OA) changes in both hip joints (Figure 1A). The OA changes were minimal in knee, ankle, foot, shoulder, elbow, hand and wrist joints (Figure 1B and C). Only an episode of transient pain in the left knee was noted as well as transient pain in the right elbow, the right shoulder and the left hand. The hip pain could not be controlled by medication, and he underwent bilateral total hip replacement at aged 57 years.

Figure 1 Radiographs of the proband aged 56 years.
A) Hip. Terminal osteoartritic (OA) changes.
B) The left knee. Mild dysplasia with flattend femoral chondyle and tibial spine.
C) The right hand. Advanced OA of the MP (metacarpo- phalangeal) and CM (carpo-metacarpal) joints.

The daughter of the proband, a 37-year-old housewife, had the bilateral knee pain; the right since the age 32 years and the left since the age 35 years, respectively. She had no symptom in other joints. Physical examination revealed no remarkable findings. Her height was 156 cm, and her weight 51 kg. The son of the proband was a 33-year-old factory worker. His height was 161 cm, and his weight 57 kg. He was found to have MED when he visited us because of the injury of the anterior cruciate ligament of the right knee. He had occasional vague pain in the right knee on climbing stairs, but had no symptom in other joints. Both individuals had no sign and symptom of myopathy. Their CK levels were normal (80 and 123 U/L). Radiographic examination identified dysplasia of the hip, knee, and the 1st and 2nd metatarsal joints in both patients (Fig. 2). Hip dysplasias were marked in the son, but minimal in the daughter, while knee changes were similar and mild.

Figure 2 Radiographs of the affected family members.
A) and C) The daughter at aged 37 years.
B) D) and E) The son at aged 33 years.
A) and B) FHip joints. Dysplasia was marked in B, but minimal in A.
C) and D) Knee joints. Dysplasias of the knee joints were both mild.

Thus, myopathy was clearly absent in our cases. We consider the association of myopathy with R718W in the JakkulaÕs case is fortuitous. Although more than 50 COMP mutations have been reported, they present MED or more severe pseudoachondroplasia (PSACH) phenotype, but not myopathy. The family 1 of the JakkulaÕs a report [1] is the only exception. COMP is expressed in skeletal muscle, but not abundant. Even the closely-situated mutation, G719D, did not present myopathy, though it showed severe PSACH phenotype.[5]

In our family, the skeletal changes were more severe in the hip joint, which is consistent with the previous assumption for the phenotype of the COMP mutation.[2,3] Hip changes in patients with COMP mutations progress with age as typically seen in PSACH. It is also true in the cases of Jakkula et al.[1] The inconsistency of their cases with the previous assumption may result from the patients' age at observation. Two affected individuals in the family did develop severe hip OA in adulthood. Alternatively, it may be another example of phenotypic variation of the same mutation.

References

1) Jakkula E, Lohiniva J, Capone A, et al. A recurrent R718W mutation in COMP results in multiple epiphyseal dysplasia with mild myopathy: clinical and pathogenetic overlap with collagen IX mutations. J Med Genet 2003; 40:942-8.

2) Unger SL, Briggs MD, Holden P, et al. Multiple epiphyseal dysplasia: radiographic abnormalities correlated with genotype. Pediatr Radiol 2001; 31:10-8.

3) Briggs MD, Chapman KL. Pseudoachondroplasia and multiple epiphyseal dysplasia: mutation review, molecular interactions, and genotype to phenotype correlations. Hum Mutat 2002; 19:465-78.

4) Mabuchi A, Manabe N, Haga N, et al. Novel types of COMP mutations and genotype-phenotype association in pseudoachondroplasia and multiple epiphyseal dysplasia. Hum Genet 2003; 112:84-90.

5) Mabuchi A, Haga N, Ikeda T, et al. A novel mutation in exon 18 of the cartilage oligomeric matrix protein gene causes a severe pseudoachondroplasia. Am J Med Genet 2001; 104:135-9.