Thomas L Ortel,1 Sang Won Shin,2 Jae Hong Seo,2 Michael J Kelley1 Genotype-Phenotype Analysis of Non-muscle Myosin Heavy Chain Mutations in Patients with May-Hegglin Anomaly Thomas L Ortel,1 Sang Won Shin,2 Jae Hong Seo,2 Michael J Kelley1 1Department of Medicine, Duke University Medical Center, and Durham VA Hospital, Durham NC, USA 2Department of Internal Medicine, Korea University Ansan Hospital, Ansan, Korea ISTH July 2001, Paris

May-Hegglin Anomaly Triad of giant platelets, thrombocytopenia, and leukocyte inclusions Absent to moderate bleeding diathesis Normal platelet function Autosomal dominant inheritance Related syndromes: Sebastian (variant inclusions) and Fechtner (features of Alport) May-Hegglin anomaly is characterized by the triad of giant platelets, thrombocytopenia, and prominent leukocyte inclusions in leukocytes vaguely similar to Dohle bodies

May-Hegglin Anomaly: Etiology Linkage analysis identified chromosome 22q12-13 as site of disease gene Non-muscle myosin heavy chain type A (MYH9) globular head (ATPase) coiled-coil rod tail Linkage analysis identified chromosome 22q12-13 as the site fo the disease gene. Subsequent positional cloning identified mutations of NMMHC-A (MYH9) gene in MHA and the related Sebastian and Fechtner syndromes. Mutations associated with MHA and Sebastian syndrome have predominantly been found in the coiled-coil rod domain and tail domain of MYH9. While Fechtner syndrome-associated muations are more common in the globular head domain containing the ATPase activity of NMMHC-A. Mutations in rod domain affect conserved residues and at least some are predicted to affect filament formation by alteration of electrostatic inter-molecule interactions. Tail truncation previously shown to affect filament formation in ?fly. * * MHA/Sebastian: Fechtner: * * * * * * * * * * * * missense ** truncation mutation (nonsense, frameshift) Hum Genet 106:557; Nat Genet 26:108; Nat Genet 26:103; Blood 97:1147; unpublished results

MHA: Methods Medical and genetic history assessment including bleeding diathesis, hearing loss, kidney dysfunction, cataract formation Affection status determined by automated CBC and review of Wright-stained peripheral blood film DNA isolated from peripheral blood used for genetic analysis of MYH9 by RFLP &/or sequencing Single CBC - various machines

MHA: Ethnicity Mutation Family # Race Ethnicity E1841K 1 Caucasian German 6 Caucasian European* 8 Caucasian Irish/English 13 Caucasian European 16 Caucasian European K Asian Korean R1933Ter 4 Caucasian Hispanic 5 Caucasian European 11 Caucasian Irish 14 Caucasian European 17 Caucasian European T1155I 3 Caucasian European Studied 12 families with history of MHA - one Korean and one Hispanic No African/black/Negro families All but one family had one of two mutations Same mutations found in diverse ethnic groups *Mixed or unknown European ethnicity

MHA: Families Unaffected At Risk Offspring Mutation Family # Gene Carriers Affected Unaffected E1841K 1 0 7 11 6 0 0 0 8 0 1 0 13 0 0 0 16 0 5 1 K 0 3 4 R1933Ter 4 0 1 3 5 0 1 2 11 0 0 0 14 0 4 0 17 0 0 0 T1155I 3 0 1 1 TOTAL 0 23 21 Among all the families, there were no unaffected mutation carriers in any family At risk individuals defined as offspring of affected individuals No significant difference between expected and observed number of affected and unaffected offspring by Chi-square test. However, family ascertainment may be biased toward families with multiple affected individuals. Therefore, can not rule out small effect on fertility or fetal survival. Prospective studies will be needed to address this possibility.

MHA: Phenotype Mutation Family # Hemorrhagic Sym Alport stigmata E1841K 1 minimal none 6 none none 8 mild none* 13 mild none 16 none none K none none R1933Ter 4 none none 5 none none 11 none none 14 none none 17 none none T1155I 3 none none Hemorrhagic symptoms were minimal or mild in 3 families with E1841K mutation while no symptoms were found in families with the other mutations. Hemorrhagic symptoms varied from easy bruisability without disability to “increased bleeding” after one of three major surgeries in one individual. None of these symptoms are clearly attributable to altered platelet number or function as a result of MHA. One individual with an E1841K mutation had had nephrotic syndrome as a child which spontaneously resolved - this was not felt to be a component of Fechtner syndrome. *one individual with h/o nephrotic syndrome - resolved

MHA: Platelet count and MPV Mutation Family # Plt, mean (range) MPV, mean (range) E1841K 1 55 (5-114) 10.4 (8.3-11.9) 6 41 9.4 8 28 (27-28) 10.5 (9.9-11) 13 13 10.6 16 38 (26-48) 11.0 (10.4-11.6) K 41 (19-68) n/d Combined 42 (5-114)(n=19) 10.6 (8.3-11.9)(n=14) R1933Ter 4 18 (17-18) 13.1 5 25 (11-39) 18.7 11 4 (2-6) 7.3 14 5 (1-15) 6.4 (4-8.8) 17 n/d n/d Combined 10.7 (1-39)(n=11) 10.4 (4-18.7)(n=5) T1155I 3 23 (3-42) 11.2 (10.8-11.5) Platelet counts were available from 19 individuals in the E1841K group, 11 in the R1933Ter group and 2 in the T1155I mutation. Two individuals in family one had platelet clumping and were not included. The single affected individual stuided in family 17 had only platelet estimates available from PBS and was likewise excluded. The mean platelet count in the E1841K mutation group was 42,000 compated to 10.7 in the 11 individuals with R1933Ter mutation. This difference was highly statistically significant with a p value of < 0.0001 by Wilcoxon test. The MPV was only slightly lower in the R1933Ter group and not statistically significantly different. However, this group had more samples in which the MPV could not be determined by the particle counters.

MHA: Platelet volume histogram Normal May-Hegglin anomaly

MHA: WBC and hemoglobin Mutation Family # WBC, mean (range) Hb, mean (range) E1841K 1 6.5 (4.1-8.1) 15.0 (13.4-17.3) 6 9.3 12.5 8 5.0 (4.1-5.8) 14.4 (12.8-16.0) 13 4.6 13.2 16 6.1 (5.1-7.2) 13.8 (12.5-15.5) K n/d n/d Combined 6.3 (4.1-9.3)(n=17) 14.3 (12.5-17.3)(n=17) R1933Ter 4 6.0 (5.5-6.4) 12 (11.8-12.2) 5 5.8 (5.0-6.5) 14.5 (13.5-15.6) 11 10.0 (9.3-10.6) 14.4 (13.9-14.9) 14 7.1 (6.1-7.7) 13.7 (13.2-15.4) 17 n/d n/d Combined 7.2 (5.0-10.6)(n=11) 13.7 (11.8-15.6)(n=11) T1155I 3 4.9-5.3 13.9-13.9 As expected, there was no significant difference in other hematologic indices, such as WBC and Hb, between the two groups of mutations

Conclusions MHA results from MYH9 mutation in diverse ethnic groups MYH9 mutations associated with MHA are highly penetrant Two MYH9 mutations account for large majority of MHA Automated platelet counts are lower in R1933Ter mutations, possibly due to larger platelet size MHA results from MYH9 mutation in diverse ethnic groups though there are no reports of MHA or MYH9 mutations in those of African descent. MYH9 mutations associated with MHA are highly penetrant - no unaffected carriers of mutant gene have been identified. While at least 8 mutations of MYH9 have been described in MHA, two MYH9 mutations account for large majority of MHA. Furthermore, there is no evidence locus heterogeneity (i.e., all families identified had some mutation in MYH9) Automated platelet counts are lower in R1933Ter mutations. Further investigation is needed (ongoing?) to determine if this is due to a difference in platelet size between the two mutations or other cause (such as artifact of different settings on particle counters)