1. 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

2. 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

3. 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Hum Genet 106:557; Nat Genet 26:108; Nat Genet 26:103; Blood 97:1147; unpublished results

4. 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

5. 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

6. MHA: Families Unaffected At Risk Offspring
Mutation Family # Gene Carriers Affected Unaffected
E1841K K
R1933Ter
T1155I
TOTAL
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.

7. 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

8. MHA: Platelet count and MPV
Mutation Family # Plt, mean (range) MPV, mean (range)
E1841K (5-114) ( )
8 28 (27-28) (9.9-11)
(26-48) ( )
K 41 (19-68) n/d
Combined 42 (5-114)(n=19) ( )(n=14)
R1933Ter (17-18) 13.1
5 25 (11-39) 18.7
11 4 (2-6) 7.3
14 5 (1-15) (4-8.8)
17 n/d n/d
Combined (1-39)(n=11) 10.4 (4-18.7)(n=5)
T1155I (3-42) ( )
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 < 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.

9. MHA: Platelet volume histogram
Normal
May-Hegglin
anomaly

10. MHA: WBC and hemoglobin
Mutation Family # WBC, mean (range) Hb, mean (range)
E1841K ( ) ( )
( ) ( )
( ) ( )
K n/d n/d
Combined ( )(n=17) 14.3 ( )(n=17)
R1933Ter ( ) 12 ( )
( ) ( )
( ) ( )
( ) ( )
17 n/d n/d
Combined ( )(n=11) 13.7 ( )(n=11)
T1155I
As expected, there was no significant difference in other hematologic indices, such as WBC and Hb, between the two groups of mutations

11. 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)